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1. Aminoglycoside tolerance in Vibrio cholerae engages translational reprogramming associated with queuosine tRNA modification

Author

Louna Fruchard, Anamaria Babosan, Andre Carvalho, Manon Lang, Blaise Li, Magalie Duchateau, Quentin Giai Gianetto, Mariette Matondo, Frederic Bonhomme, Isabelle Hatin, Hugo Arbes, Céline Fabret, Enora Corler, Guillaume Sanchez, Virginie Marchand, Yuri Motorin, Olivier Namy, Valérie de Crécy-Lagard, Didier Mazel, and Zeynep Baharoglu

Subjects
Vibrio cholerae, epitranscriptome, antibiotic stress, tRNA modification, queuosine, post-transcriptional regulation, Medicine, Science, Biology (General), QH301-705.5
Abstract

Tgt is the enzyme modifying the guanine (G) in tRNAs with GUN anticodon to queuosine (Q). tgt is required for optimal growth of Vibrio cholerae in the presence of sub-lethal aminoglycoside concentrations. We further explored here the role of the Q34 in the efficiency of codon decoding upon tobramycin exposure. We characterized its impact on the overall bacterial proteome, and elucidated the molecular mechanisms underlying the effects of Q34 modification in antibiotic translational stress response. Using molecular reporters, we showed that Q34 impacts the efficiency of decoding at tyrosine TAT and TAC codons. Proteomics analyses revealed that the anti-SoxR factor RsxA is better translated in the absence of tgt. RsxA displays a codon bias toward tyrosine TAT and overabundance of RsxA leads to decreased expression of genes belonging to SoxR oxidative stress regulon. We also identified conditions that regulate tgt expression. We propose that regulation of Q34 modification in response to environmental cues leads to translational reprogramming of transcripts bearing a biased tyrosine codon usage. In silico analysis further identified candidate genes which could be subject to such translational regulation, among which DNA repair factors. Such transcripts, fitting the definition of modification tunable transcripts, are central in the bacterial response to antibiotics.

Published
2025
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2. Mtfp1 ablation enhances mitochondrial respiration and protects against hepatic steatosis

Author

Cecilia Patitucci, Juan Diego Hernández-Camacho, Elodie Vimont, Sonny Yde, Thomas Cokelaer, Thibault Chaze, Quentin Giai Gianetto, Mariette Matondo, Anastasia Gazi, Ivan Nemazanyy, David A. Stroud, Daniella H. Hock, Erminia Donnarumma, and Timothy Wai

Subjects
Science
Abstract

Abstract Hepatic steatosis is the result of imbalanced nutrient delivery and metabolism in the liver and is the first hallmark of Metabolic dysfunction-associated steatotic liver disease (MASLD). MASLD is the most common chronic liver disease and involves the accumulation of excess lipids in hepatocytes, inflammation, and cancer. Mitochondria play central roles in liver metabolism yet the specific mitochondrial functions causally linked to MASLD remain unclear. Here, we identify Mitochondrial Fission Process 1 protein (MTFP1) as a key regulator of mitochondrial and metabolic activity in the liver. Deletion of Mtfp1 in hepatocytes is physiologically benign in mice yet leads to the upregulation of oxidative phosphorylation (OXPHOS) activity and mitochondrial respiration, independently of mitochondrial biogenesis. Consequently, liver-specific knockout mice are protected against high fat diet-induced steatosis and metabolic dysregulation. Additionally, Mtfp1 deletion inhibits mitochondrial permeability transition pore opening in hepatocytes, conferring protection against apoptotic liver damage in vivo and ex vivo. Our work uncovers additional functions of MTFP1 in the liver, positioning this gene as an unexpected regulator of OXPHOS and a therapeutic candidate for MASLD.

Published
2023
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3. Contribution of MALDI‐TOF mass spectrometry and machine learning including deep learning techniques for the detection of virulence factors of Clostridioides difficile strains

Author

Alexandre Godmer, Quentin Giai Gianetto, Killian Le Neindre, Valentine Latapy, Mathilda Bastide, Muriel Ehmig, Valérie Lalande, Nicolas Veziris, Alexandra Aubry, Frédéric Barbut, and Catherine Eckert

Subjects
Biotechnology, TP248.13-248.65
Abstract

Abstract Clostridioides difficile (CD) infections are defined by toxins A (TcdA) and B (TcdB) along with the binary toxin (CDT). The emergence of the ‘hypervirulent’ (Hv) strain PR 027, along with PR 176 and 181, two decades ago, reshaped CD infection epidemiology in Europe. This study assessed MALDI‐TOF mass spectrometry (MALDI‐TOF MS) combined with machine learning (ML) and Deep Learning (DL) to identify toxigenic strains (producing TcdA, TcdB with or without CDT) and Hv strains. In total, 201 CD strains were analysed, comprising 151 toxigenic (24 ToxA+B+CDT+, 22 ToxA+B+CDT+ Hv+ and 105 ToxA+B+CDT−) and 50 non‐toxigenic (ToxA−B−) strains. The DL‐based classifier exhibited a 0.95 negative predictive value for excluding ToxA−B− strains, showcasing accuracy in identifying this strain category. Sensitivity in correctly identifying ToxA+B+CDT− strains ranged from 0.68 to 0.91. Additionally, all classifiers consistently demonstrated high specificity (>0.96) in detecting ToxA+B+CDT+ strains. The classifiers' performances for Hv strain detection were linked to high specificity (≥0.96). This study highlights MALDI‐TOF MS enhanced by ML techniques as a rapid and cost‐effective tool for identifying CD strain virulence factors. Our results brought a proof‐of‐concept concerning the ability of MALDI‐TOF MS coupled with ML techniques to detect virulence factor and potentially improve the outbreak's management.

Published
2024
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4. Using a multiomics approach to unravel a septic shock specific signature in skeletal muscle

Author

Baptiste Duceau, Michael Blatzer, Jean Bardon, Thibault Chaze, Quentin Giai Gianetto, Florence Castelli, François Fenaille, Lucie Duarte, Thomas Lescot, Christophe Tresallet, Bruno Riou, Mariette Matondo, Olivier Langeron, Pierre Rocheteau, Fabrice Chrétien, and Adrien Bouglé

Subjects
Medicine, Science
Abstract

Abstract Sepsis is defined as a dysregulated host response to infection leading to organs failure. Among them, sepsis induces skeletal muscle (SM) alterations that contribute to acquired-weakness in critically ill patients. Proteomics and metabolomics could unravel biological mechanisms in sepsis-related organ dysfunction. Our objective was to characterize a distinctive signature of septic shock in human SM by using an integrative multi-omics approach. Muscle biopsies were obtained as part of a multicenter non-interventional prospective study. Study population included patients in septic shock (S group, with intra-abdominal source of sepsis) and two critically ill control populations: cardiogenic shock (C group) and brain dead (BD group). The proteins and metabolites were extracted and analyzed by High-Performance Liquid Chromatography-coupled to tandem Mass Spectrometry, respectively. Fifty patients were included, 19 for the S group (53% male, 64 ± 17 years, SAPS II 45 ± 14), 12 for the C group (75% male, 63 ± 4 years, SAPS II 43 ± 15), 19 for the BD group (63% male, 58 ± 10 years, SAPS II 58 ± 9). Biopsies were performed in median 3 days [interquartile range 1–4]) after intensive care unit admission. Respectively 31 patients and 40 patients were included in the proteomics and metabolomics analyses of 2264 proteins and 259 annotated metabolites. Enrichment analysis revealed that mitochondrial pathways were significantly decreased in the S group at protein level: oxidative phosphorylation (adjusted p = 0.008); branched chained amino acids degradation (adjusted p = 0.005); citrate cycle (adjusted p = 0.005); ketone body metabolism (adjusted p = 0.003) or fatty acid degradation (adjusted p = 0.008). Metabolic reprogramming was also suggested (i) by the differential abundance of the peroxisome proliferator-activated receptors signaling pathway (adjusted p = 0.007), and (ii) by the accumulation of fatty acids like octanedioic acid dimethyl or hydroxydecanoic. Increased polyamines and depletion of mitochondrial thioredoxin or mitochondrial peroxiredoxin indicated a high level of oxidative stress in the S group. Coordinated alterations in the proteomic and metabolomic profiles reveal a septic shock signature in SM, highlighting a global impairment of mitochondria-related metabolic pathways, the depletion of antioxidant capacities, and a metabolic shift towards lipid accumulation. ClinicalTrial registration: NCT02789995. Date of first registration 03/06/2016.

Published
2022
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5. Mitochondrial Fission Process 1 controls inner membrane integrity and protects against heart failure

Author

Erminia Donnarumma, Michael Kohlhaas, Elodie Vimont, Etienne Kornobis, Thibault Chaze, Quentin Giai Gianetto, Mariette Matondo, Maryse Moya-Nilges, Christoph Maack, and Timothy Wai

Subjects
Science
Abstract

Mitochondria power the beating heart. Here, Donnarumma et al. show that loss of the inner mitochondrial membrane protein MTFP1 in cardiomyocytes reduces bioenergetic efficiency and cell death resistance leading to heart failure in mice.

Published
2022
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6. A time-resolved multi-omics atlas of Acanthamoeba castellanii encystment

Author

Clément Bernard, Marie Locard-Paulet, Cyril Noël, Magalie Duchateau, Quentin Giai Gianetto, Bouziane Moumen, Thomas Rattei, Yann Hechard, Lars Juhl Jensen, Mariette Matondo, and Ascel Samba-Louaka

Subjects
Science
Abstract

Encystment is a process that allows free-living and parasitic amoebae to survive in unfavorable environments. Here, Bernard et al. provide detailed insights into the early stages of encystment of Acanthamoeba castellanii by integrating RNA-Seq, proteomics and phosphoproteomics data sets.

Published
2022
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7. Loss of CorA, the primary magnesium transporter of Salmonella, is alleviated by MgtA and PhoP-dependent compensatory mechanisms.

Author

Selma Metaane, Véronique Monteil, Thibaut Douché, Quentin Giai Gianetto, Mariette Matondo, Corinne Maufrais, and Françoise Norel

Subjects
Medicine, Science
Abstract

In many Gram-negative bacteria, the stress sigma factor of RNA polymerase, σS/RpoS, remodels global gene expression to reshape the physiology of stationary phase cells and ensure their survival under non-optimal growth conditions. In the foodborne pathogen Salmonella enterica serovar Typhimurium, σS is also required for biofilm formation and virulence. We have recently shown that a ΔrpoS mutation decreases the magnesium content and expression level of the housekeeping Mg2+-transporter CorA in stationary phase Salmonella. The other two Mg2+-transporters of Salmonella are encoded by the PhoP-activated mgtA and mgtB genes and are expressed under magnesium starvation. The σS control of corA prompted us to evaluate the impact of CorA in stationary phase Salmonella cells, by using global and analytical proteomic analyses and physiological assays. The ΔcorA mutation conferred a competitive disadvantage to exit from stationary phase, and slightly impaired motility, but had no effect on total and free cellular magnesium contents. In contrast to the wild-type strain, the ΔcorA mutant produced MgtA, but not MgtB, in the presence of high extracellular magnesium concentration. Under these conditions, MgtA production in the ΔcorA mutant did not require PhoP. Consistently, a ΔmgtA, but not a ΔphoP, mutation slightly reduced the magnesium content of the ΔcorA mutant. Synthetic phenotypes were observed when the ΔphoP and ΔcorA mutations were combined, including a strong reduction in growth and motility, independently of the extracellular magnesium concentration. The abundance of several proteins involved in flagella formation, chemotaxis and secretion was lowered by the ΔcorA and ΔphoP mutations in combination, but not alone. These findings unravel the importance of PhoP-dependent functions in the absence of CorA when magnesium is sufficient. Altogether, our data pinpoint a regulatory network, where the absence of CorA is sensed by the cell and compensated by MgtA and PhoP- dependent mechanisms.

Published
2023
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8. Bacterial Membrane Vesicles as a Novel Strategy for Extrusion of Antimicrobial Bismuth Drug in Helicobacter pylori

Author

Sumith Kumar, Christine Schmitt, Olivier Gorgette, Martial Marbouty, Magalie Duchateau, Quentin Giai Gianetto, Mariette Matondo, Jean-Michel Guigner, and Hilde De Reuse

Subjects
Helicobacter pylori, bismuth, membrane vesicles, antibiotic resistance, polyphosphate granules, chromosome conformation capture, Microbiology, QR1-502
Abstract

ABSTRACT Bacterial antibiotic resistance is a major threat to human health. A combination of antibiotics with metals is among the proposed alternative treatments. Only one such combination is successfully used in clinics; it associates antibiotics with the metal bismuth to treat infections by Helicobacter pylori. This bacterial pathogen colonizes the human stomach and is associated with gastric cancer, killing 800,000 individuals yearly. The effect of bismuth in H. pylori treatment is not well understood in particular for sublethal doses such as those measured in the plasma of treated patients. We addressed this question and observed that bismuth induces the formation of homogeneously sized membrane vesicles (MVs) with unique protein cargo content enriched in bismuth-binding proteins, as shown by quantitative proteomics. Purified MVs of bismuth-exposed bacteria were strongly enriched in bismuth as measured by inductively coupled plasma optical emission spectrometry (ICP-OES), unlike bacterial cells from which they originate. Thus, our results revealed a novel function of MVs in bismuth detoxification, where secreted MVs act as tool to discard bismuth from the bacteria. Bismuth also induces the formation of intracellular polyphosphate granules that are associated with changes in nucleoid structure. Nucleoid compaction in response to bismuth was established by immunogold electron microscopy and refined by the first chromosome conformation capture (Hi-C) analysis of H. pylori. Our results reveal that even low doses of bismuth induce profound changes in H. pylori physiology and highlight a novel defense mechanism that involves MV-mediated bismuth extrusion from the bacteria and a probable local DNA protective response where polyphosphate granules are associated with nucleoid compaction. IMPORTANCE Bacterial resistance to antibiotics is a major threat to human health. Treatments combining antibiotics with metals were proposed to circumvent this hurdle. Only one such combination is successfully used in clinics associating antibiotics with the metal bismuth to treat infections by the human pathogen Helicobacter pylori. H. pylori causes 800,000 deaths by gastric cancer yearly. How bismuth impacts H. pylori and its response to this toxic metal were ill defined. We discovered that upon bismuth exposure, H. pylori secretes membrane vesicles that are enriched in bismuth. Bismuth also induces the formation of intracellular polyphosphate granules associated with compaction of the chromosome. Upon bismuth exposure, H. pylori displays both defense and protection mechanisms, with bismuth extrusion by vesicles and shielding of the chromosome.

Published
2022
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9. The Flemmingsome reveals an ESCRT-to-membrane coupling via ALIX/syntenin/syndecan-4 required for completion of cytokinesis

Author

Cyril Addi, Adrien Presle, Stéphane Frémont, Frédérique Cuvelier, Murielle Rocancourt, Florine Milin, Sandrine Schmutz, Julia Chamot-Rooke, Thibaut Douché, Magalie Duchateau, Quentin Giai Gianetto, Audrey Salles, Hervé Ménager, Mariette Matondo, Pascale Zimmermann, Neetu Gupta-Rossi, and Arnaud Echard

Subjects
Science
Abstract

ESCRT filaments drive the final abscission between two daughter cells but how they physically interact with the membrane is unclear. Using proteomics, the authors show that syndecan-4/syntenin/ALIX couples the ESCRT machinery to the abscission site and thus promotes efficient abscission.

Published
2020
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10. RNase E and HupB dynamics foster mycobacterial cell homeostasis and fitness

Author

Anna Griego, Thibaut Douché, Quentin Giai Gianetto, Mariette Matondo, and Giulia Manina

Subjects
Environmental science, Ecology, Biological sciences, Microbiology, Science
Abstract

Summary: RNA turnover is a primary source of gene expression variation, in turn promoting cellular adaptation. Mycobacteria leverage reversible mRNA stabilization to endure hostile conditions. Although RNase E is essential for RNA turnover in several species, its role in mycobacterial single-cell physiology and functional phenotypic diversification remains unexplored. Here, by integrating live-single-cell and quantitative-mass-spectrometry approaches, we show that RNase E forms dynamic foci, which are associated with cellular homeostasis and fate, and we discover a versatile molecular interactome. We show a likely interaction between RNase E and the nucleoid-associated protein HupB, which is particularly pronounced during drug treatment and infection, where phenotypic diversity increases. Disruption of RNase E expression affects HupB levels, impairing Mycobacterium tuberculosis growth homeostasis during treatment, intracellular replication, and host spread. Our work lays the foundation for targeting the RNase E and its partner HupB, aiming to undermine M. tuberculosis cellular balance, diversification capacity, and persistence.

Published
2022
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11. Impact of the gut microbiota on the m6A epitranscriptome of mouse cecum and liver

Author

Sabrina Jabs, Anne Biton, Christophe Bécavin, Marie-Anne Nahori, Amine Ghozlane, Alessandro Pagliuso, Giulia Spanò, Vincent Guérineau, David Touboul, Quentin Giai Gianetto, Thibault Chaze, Mariette Matondo, Marie-Agnès Dillies, and Pascale Cossart

Subjects
Science
Abstract

The intestinal microbiota modulates host physiology and gene expression via unclear mechanisms. Here, Jabs et al. show that variations in the gut microbiota correlate with N6-methyladenosine modifications of host mRNAs in the cecum and liver of mice.

Published
2020
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12. Ser/Thr Kinase-Dependent Phosphorylation of the Peptidoglycan Hydrolase CwlA Controls Its Export and Modulates Cell Division in Clostridioides difficile

Author

Transito Garcia-Garcia, Sandrine Poncet, Elodie Cuenot, Thibaut Douché, Quentin Giai Gianetto, Johann Peltier, Pascal Courtin, Marie-Pierre Chapot-Chartier, Mariette Matondo, Bruno Dupuy, Thomas Candela, and Isabelle Martin-Verstraete

Subjects
Microbiology, QR1-502
Abstract

Bacterial cells are encased in a PG exoskeleton that helps to maintain cell shape and confers physical protection. To allow bacterial growth and cell separation, PG needs to be continuously remodeled by hydrolytic enzymes that cleave PG at critical sites.

Published
2021
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13. Proteomic Analysis of Humoral Immune Components in Bronchoalveolar Lavage of Patients Infected or Colonized by Aspergillus fumigatus

Author

Sarah Dellière, Magalie Duchateau, Sarah Sze Wah Wong, Quentin Giai Gianetto, Hélène Guegan, Mariette Matondo, Jean-Pierre Gangneux, and Vishukumar Aimanianda

Subjects
Aspergillus fumigatus, aspergillosis, bronchoalveolar lavage, proteomics, humoral immunity, complement system, Immunologic diseases. Allergy, RC581-607
Abstract

Humoral immune components have been individually studied in the context of interaction of host with Aspergillus fumigatus, a major airborne fungal pathogen. However, a global view of the multitude and complex nature of humoral immune components is needed to bring new insight into host-Aspergillus interaction. Therefore, we undertook comparative proteomic analysis of the bronchoalveolar lavage fluid collected from individuals infected or colonized with A. fumigatus versus controls, to identify those alveolar humoral components affected upon A. fumigatus infection. Complement proteins C1q, C8 beta-chain, factor-H, ficolin-1, ficolin-2, mannan binding lectin serine peptidase 2, pentraxin-3 and the surfactant protein-D were identified as the major humoral immune components affected by A. fumigatus infection and colonization. Based on this observation, we hypothesize that crosstalk between these humoral components is essential during host-Aspergillus interaction giving new specific leads to study for better understanding the pathogenesis. Furthermore, the affected humoral components could be potential diagnostic markers of A. fumigatus infection or colonization.

Published
2021
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14. CYP450 core involvement in multiple resistance strains of Aedes aegypti from French Guiana highlighted by proteomics, molecular and biochemical studies.

Author

Yanouk Epelboin, Lanjiao Wang, Quentin Giai Gianetto, Valérie Choumet, Pascal Gaborit, Jean Issaly, Amandine Guidez, Thibaut Douché, Thibault Chaze, Mariette Matondo, and Isabelle Dusfour

Subjects
Medicine, Science
Abstract

Insecticide resistance is a worldwide threat for vector control around the world, and Aedes aegypti, the main vector of several arboviruses, is a particular concern. To better understand the mechanisms of resistance, four isofemale strains originally from French Guiana were isolated and analysed using combined approaches. The activity of detoxification enzymes involved in insecticide resistance was assayed, and mutations located at positions 1016 and 1534 of the sodium voltage-gated channel gene, which have been associated with pyrethroid resistance in Aedes aegypti populations in Latin America, were monitored. Resistance to other insecticide families (organophosphates and carbamates) was evaluated. A large-scale proteomic analysis was performed to identify proteins involved in insecticide resistance. Our results revealed a metabolic resistance and resistance associated with a mutation of the sodium voltage-gated channel gene at position 1016. Metabolic resistance was mediated through an increase of esterase activity in most strains but also through the shifts in the abundance of several cytochrome P450 (CYP450s). Overall, resistance to deltamethrin was linked in the isofemale strains to resistance to other class of insecticides, suggesting that cross- and multiple resistance occur through selection of mechanisms of metabolic resistance. These results give some insights into resistance to deltamethrin and into multiple resistance phenomena in populations of Ae. aegypti.

Published
2021
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15. Differentiation-dependent susceptibility of human muscle cells to Zika virus infection.

Author

Vincent Legros, Patricia Jeannin, Julien Burlaud-Gaillard, Thibault Chaze, Quentin Giai Gianetto, Gillian Butler-Browne, Vincent Mouly, Jim Zoladek, Philippe V Afonso, Mariela-Natacha Gonzàlez, Mariette Matondo, Ingo Riederer, Philippe Roingeard, Antoine Gessain, Valérie Choumet, and Pierre-Emmanuel Ceccaldi

Subjects
Arctic medicine. Tropical medicine, RC955-962, Public aspects of medicine, RA1-1270
Abstract

Muscle cells are potential targets of many arboviruses, such as Ross River, Dengue, Sindbis, and chikungunya viruses, that may be involved in the physiopathological course of the infection. During the recent outbreak of Zika virus (ZIKV), myalgia was one of the most frequently reported symptoms. We investigated the susceptibility of human muscle cells to ZIKV infection. Using an in vitro model of human primary myoblasts that can be differentiated into myotubes, we found that myoblasts can be productively infected by ZIKV. In contrast, myotubes were shown to be resistant to ZIKV infection, suggesting a differentiation-dependent susceptibility. Infection was accompanied by a caspase-independent cytopathic effect, associated with paraptosis-like cytoplasmic vacuolization. Proteomic profiling was performed 24h and 48h post-infection in cells infected with two different isolates. Proteome changes indicate that ZIKV infection induces an upregulation of proteins involved in the activation of the Interferon type I pathway, and a downregulation of protein synthesis. This work constitutes the first observation of primary human muscle cells susceptibility to ZIKV infection, and differentiation-dependent restriction of infection from myoblasts to myotubes. Since myoblasts constitute the reservoir of stem cells involved in reparation/regeneration in muscle tissue, the infection of muscle cells and the viral-induced alterations observed here could have consequences in ZIKV infection pathogenesis.

Published
2020
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16. Phosphorylation-Dependent Assembly of a 14-3-3 Mediated Signaling Complex during Red Blood Cell Invasion by Plasmodium falciparum Merozoites

Author

Kunal R. More, Inderjeet Kaur, Quentin Giai Gianetto, Brandon M. Invergo, Thibault Chaze, Ravi Jain, Christéle Huon, Petra Gutenbrunner, Hendrik Weisser, Mariette Matondo, Jyoti S. Choudhary, Gordon Langsley, Shailja Singh, and Chetan E. Chitnis

Subjects
host cell invasion, host-parasite interaction, malaria, signaling, Microbiology, QR1-502
Abstract

ABSTRACT Red blood cell (RBC) invasion by Plasmodium merozoites requires multiple steps that are regulated by signaling pathways. Exposure of P. falciparum merozoites to the physiological signal of low K+, as found in blood plasma, leads to a rise in cytosolic Ca2+, which mediates microneme secretion, motility, and invasion. We have used global phosphoproteomic analysis of merozoites to identify signaling pathways that are activated during invasion. Using quantitative phosphoproteomics, we found 394 protein phosphorylation site changes in merozoites subjected to different ionic environments (high K+/low K+), 143 of which were Ca2+ dependent. These included a number of signaling proteins such as catalytic and regulatory subunits of protein kinase A (PfPKAc and PfPKAr) and calcium-dependent protein kinase 1 (PfCDPK1). Proteins of the 14-3-3 family interact with phosphorylated target proteins to assemble signaling complexes. Here, using coimmunoprecipitation and gel filtration chromatography, we demonstrate that Pf14-3-3I binds phosphorylated PfPKAr and PfCDPK1 to mediate the assembly of a multiprotein complex in P. falciparum merozoites. A phospho-peptide, P1, based on the Ca2+-dependent phosphosites of PKAr, binds Pf14-3-3I and disrupts assembly of the Pf14-3-3I-mediated multiprotein complex. Disruption of the multiprotein complex with P1 inhibits microneme secretion and RBC invasion. This study thus identifies a novel signaling complex that plays a key role in merozoite invasion of RBCs. Disruption of this signaling complex could serve as a novel approach to inhibit blood-stage growth of malaria parasites. IMPORTANCE Invasion of red blood cells (RBCs) by Plasmodium falciparum merozoites is a complex process that is regulated by intricate signaling pathways. Here, we used phosphoproteomic profiling to identify the key proteins involved in signaling events during invasion. We found changes in the phosphorylation of various merozoite proteins, including multiple kinases previously implicated in the process of invasion. We also found that a phosphorylation-dependent multiprotein complex including signaling kinases assembles during the process of invasion. Disruption of this multiprotein complex impairs merozoite invasion of RBCs, providing a novel approach for the development of inhibitors to block the growth of blood-stage malaria parasites.

Published
2020
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17. Shigella hijacks the exocyst to cluster macropinosomes for efficient vacuolar escape.

Author

Yuen-Yan Chang, Virginie Stévenin, Magalie Duchateau, Quentin Giai Gianetto, Veronique Hourdel, Cristina Dias Rodrigues, Mariette Matondo, Norbert Reiling, and Jost Enninga

Subjects
Immunologic diseases. Allergy, RC581-607, Biology (General), QH301-705.5
Abstract

Shigella flexneri invades host cells by entering within a bacteria-containing vacuole (BCV). In order to establish its niche in the host cytosol, the bacterium ruptures its BCV. Contacts between S. flexneri BCV and infection-associated macropinosomes (IAMs) formed in situ have been reported to enhance BCV disintegration. The mechanism underlying S. flexneri vacuolar escape remains however obscure. To decipher the molecular mechanism priming the communication between the IAMs and S. flexneri BCV, we performed mass spectrometry-based analysis of the magnetically purified IAMs from S. flexneri-infected cells. While proteins involved in host recycling and exocytic pathways were significantly enriched at the IAMs, we demonstrate more precisely that the S. flexneri type III effector protein IpgD mediates the recruitment of the exocyst to the IAMs through the Rab8/Rab11 pathway. This recruitment results in IAM clustering around S. flexneri BCV. More importantly, we reveal that IAM clustering subsequently facilitates an IAM-mediated unwrapping of the ruptured vacuole membranes from S. flexneri, enabling the naked bacterium to be ready for intercellular spread via actin-based motility. Taken together, our work untangles the molecular cascade of S. flexneri-driven host trafficking subversion at IAMs to develop its cytosolic lifestyle, a crucial step en route for infection progression at cellular and tissue level.

Published
2020
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18. Proteomic analysis of plasma extracellular vesicles reveals mitochondrial stress upon HTLV-1 infection

Author

Patricia Jeannin, Thibault Chaze, Quentin Giai Gianetto, Mariette Matondo, Olivier Gout, Antoine Gessain, and Philippe V. Afonso

Subjects
Medicine, Science
Abstract

Abstract Extracellular vesicles (EVs) can participate in intercellular communication and pathogenesis. EVs contain many cargos, including proteins, and the composition of EVs differs between cell-types and activation levels. Thus, plasma EVs can be used as a biomarker of systemic response to infection and/or disease progression. In this study, we aimed at describing alterations in the protein content of plasma EVs upon infection with the human T-lymphotropic retrovirus type 1 (HTLV-1). HTLV-1 is the etiological agent of a lymphoproliferative disease (ATL) and a series of inflammatory diseases, including a neurodegenerative inflammatory disease (HAM/TSP). We found that plasma EVs are more abundant and smaller in HTLV-1 asymptomatic carriers or HAM/TSP patients when compared to uninfected healthy donors. Moreover, EVs from HTLV-1 infected donors contain markers of metabolic and mitochondrial stress.

Published
2018
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19. Dynamic Growth and Shrinkage of the Salmonella-Containing Vacuole Determines the Intracellular Pathogen Niche

Author

Virginie Stévenin, Yuen-Yan Chang, Yoann Le Toquin, Magalie Duchateau, Quentin Giai Gianetto, Chak Hon Luk, Audrey Salles, Victoria Sohst, Mariette Matondo, Norbert Reiling, and Jost Enninga

Subjects
Biology (General), QH301-705.5
Abstract

Summary: Salmonella is a human and animal pathogen that causes gastro-enteric diseases. The key to Salmonella infection is its entry into intestinal epithelial cells, where the bacterium resides within a Salmonella-containing vacuole (SCV). Salmonella entry also induces the formation of empty macropinosomes, distinct from the SCV, in the vicinity of the entering bacteria. A few minutes after its formation, the SCV increases in size through fusions with the surrounding macropinosomes. Salmonella also induces membrane tubules that emanate from the SCV and lead to SCV shrinkage. Here, we show that these antipodal events are utilized by Salmonella to either establish a vacuolar niche or to be released into the cytosol by SCV rupture. We identify the molecular machinery underlying dynamic SCV growth and shrinkage. In particular, the SNARE proteins SNAP25 and STX4 participate in SCV inflation by fusion with macropinosomes. Thus, host compartment size control emerges as a pathogen strategy for intracellular niche regulation. : When infecting epithelial cells, Salmonella can reside and replicate within vacuolar or cytosolic niches. These co-existing lifestyles provide alternative survival strategies for the bacteria. Stévenin et al. show that the balance between dynamic growth and shrinkage of the Salmonella-containing vacuole determines vacuolar maintenance or rupture and controls the pathogen niche. Keywords: Salmonella enterica serovar Typhimurium, membrane trafficking, vesicle fusion, membrane rupture, compartmental size control, Salmonella-containing vacuole, macropinosome, SNARE, spacious vacuole-associated tubules (SVAT), magnetic extraction, proteomics

Published
2019
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20. Assembly and disassembly of Aspergillus fumigatus conidial rodlets

Author

Isabel Valsecchi, Jennifer I. Lai, Emmanuel Stephen-Victor, Ariane Pillé, Audrey Beaussart, Victor Lo, Chi L.L. Pham, Vishukumar Aimanianda, Ann H. Kwan, Magalie Duchateau, Quentin Giai Gianetto, Mariette Matondo, Melanie Lehoux, Donald C. Sheppard, Yves F. Dufrene, Jagadeesh Bayry, J. Iñaki Guijarro, Margaret Sunde, and Jean-Paul Latgé

Subjects
Cytology, QH573-671
Abstract

The rodlet structure present on the Aspergillus fumigatus conidial surface hides conidia from immune recognition. In spite of the essential biological role of the rodlets, the molecular basis for their self-assembly and disaggregation is not known. Analysis of the soluble forms of conidia-extracted and recombinant RodA by NMR spectroscopy has indicated the importance of disulfide bonds and identified two dynamic regions as likely candidates for conformational change and intermolecular interactions during conversion of RodA into the amyloid rodlet structure. Point mutations introduced into the RODA sequence confirmed that (1) mutation of a single cysteine was sufficient to block rodlet formation on the conidial surface and (2) both presumed amyloidogenic regions were needed for proper rodlet assembly. Mutations in the two putative amyloidogenic regions retarded and disturbed, but did not completely inhibit, the formation of the rodlets in vitro and on the conidial surface. Even in a disturbed form, the presence of rodlets on the surface of the conidia was sufficient to immunosilence the conidium. However, in contrast to the parental conidia, long exposure of mutant conidia lacking disulfide bridges within RodA or expressing RodA carrying the double (I115S/I146G) mutation activated dendritic cells with the subsequent secretion of proinflammatory cytokines. The immune reactivity of the RodA mutant conidia was not due to a modification in the RodA structure, but to the exposure of different pathogen-associated molecular patterns on the surface as a result of the modification of the rodlet surface layer. The full degradation of the rodlet layer, which occurs during early germination, is due to a complex array of cell wall bound proteases. As reported earlier, this loss of the rodlet layer lead to a strong anti-fumigatus host immune response in mouse lungs. Keywords: Aspergillus fumigatus, Hydrophobins, Rodlets, RodA, Amyloids

Published
2019
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21. RadD Contributes to R-Loop Avoidance in Sub-MIC Tobramycin

Author

Veronica Negro, Evelyne Krin, Sebastian Aguilar Pierlé, Thibault Chaze, Quentin Giai Gianetto, Sean P. Kennedy, Mariette Matondo, Didier Mazel, and Zeynep Baharoglu

Subjects
DNA repair, R-loop, antibiotic resistance, Microbiology, QR1-502
Abstract

ABSTRACT We have previously identified Vibrio cholerae mutants in which the stress response to subinhibitory concentrations of aminoglycoside is altered. One gene identified, VC1636, encodes a putative DNA/RNA helicase, recently named RadD in Escherichia coli. Here we combined extensive genetic characterization and high-throughput approaches in order to identify partners and molecular mechanisms involving RadD. We show that double-strand DNA breaks (DSBs) are formed upon subinhibitory tobramycin treatment in the absence of radD and recBCD and that formation of these DSBs can be overcome by RNase H1 overexpression. Loss of RNase H1, or of the transcription-translation coupling factor EF-P, is lethal in the radD deletion mutant. We propose that R-loops are formed upon sublethal aminoglycoside treatment, leading to the formation of DSBs that can be repaired by the RecBCD homologous recombination pathway, and that RadD counteracts such R-loop accumulation. We discuss how R-loops that can occur upon translation-transcription uncoupling could be the link between tobramycin treatment and DNA break formation. IMPORTANCE Bacteria frequently encounter low concentrations of antibiotics. Active antibiotics are commonly detected in soil and water at concentrations much below lethal concentration. Although sub-MICs of antibiotics do not kill bacteria, they can have a major impact on bacterial populations by contributing to the development of antibiotic resistance through mutations in originally sensitive bacteria or acquisition of DNA from resistant bacteria. It was shown that concentrations as low as 100-fold below the MIC can actually lead to the selection of antibiotic-resistant cells. We seek to understand how bacterial cells react to such antibiotic concentrations using E. coli, the Gram-negative bacterial paradigm, and V. cholerae, the causative agent of cholera. Our findings shed light on the processes triggered at the DNA level by antibiotics targeting translation, how damage occurs, and what the bacterial strategies are to respond to such DNA damage.

Published
2019
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23. The RBPome of influenza A virus mRNA reveals a role for TDP-43 in viral replication

Author

Maud Dupont, Tim Krischuns, Quentin Giai-Gianetto, Sylvain Paisant, Stefano Bonazza, Jean-Baptiste Brault, Thibaut Douché, Joel I Perez-Perri, Matthias W Hentze, Stephen Cusack, Mariette Matondo, Catherine Isel, David G Courtney, Nadia Naffakh, Biologie des ARN et virus influenza - RNA Biology of Influenza Virus (CNRS-UMR3569), Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS)-Université Paris Cité (UPCité), Plateforme de Protéomique / Proteomics platform, Université Paris Cité (UPCité)-Spectrométrie de Masse pour la Biologie – Mass Spectrometry for Biology (UTechS MSBio), Institut Pasteur [Paris] (IP)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS)-Université Paris Cité (UPCité)-Institut Pasteur [Paris] (IP)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS), Hub Bioinformatique et Biostatistique - Bioinformatics and Biostatistics HUB, Institut Pasteur [Paris] (IP)-Université Paris Cité (UPCité), Wellcome-Wolfson Institute for Experimental Medicine, Queen's University [Belfast] (QUB), European Molecular Biology Laboratory [Heidelberg] (EMBL), European Molecular Biology Laboratory [Grenoble] (EMBL), This work was funded by the Agence Nationale de la Recherche [ANR-18-CE18-0028 to SC and NN, ANR-10-LABX-62IBEID to MM and NN, ANR-21-CE35-0007 to MM], the Marie-Skłodowska Curie Global Fellowship [MSCA-IF-GF:747810 to DGC], and the European Research Council Fellowship [PTFLU 949506 to DGC and SB]. Funding for open access charges [Agence Nationale de la Recherche/ANR-10-LABX-62IBEID], We would like to thank Dr. Feng Zhang (Broad Institute, Cambridge, USA), Dr. Mikhael Matrosovich (Philipps Universität, Marburg, Germany), Dr. Pierre-Olivier Vidalain (CIRI, Lyon), Dr. Daniel Marc (INRAE, Nouzilly, France), Dr. Caroline Demeret, Dr. Sandie Munier and the National Reference Center for Respiratory Viruses (all at Institut Pasteur, Paris, France) and for sharing reagents used in this work. We would like to thank Yves Jacob, Anastasia Komarova and Valérie Najburg (Institut Pasteur, Paris, France) for helpful discussions. Some figures were created with BioRender.com, ANR-10-LABX-0062,IBEID,Integrative Biology of Emerging Infectious Diseases(2010), and ANR-21-CE35-0007,PureMagRupture,Un flux de travail de la gMEP (Genetic magnetic-extraction-proteomics) pour comprendre le lien entre la rupture vacuolaire de Shigella et l'autophagie(2021)

Subjects
[SDV]Life Sciences [q-bio]
Abstract

Recent technical advances have significantly improved our understanding of the RNA-binding protein (RBP) repertoire present within eukaryotic cells, with a particular focus on the RBPs that interact with cellular polyadenylated mRNAs. However, recent studies utilising the same technologies have begun to tease apart the RBP interactome of viral mRNAs, notably SARS-CoV-2, revealing both similarities and differences between the RBP profiles of viral and cellular mRNAs. Herein, we comprehensively identified the RBPs that associate with the NP mRNA of an influenza A virus. Moreover, we provide evidence that the viral polymerase is essential for the recruitment of RPBs to viral mRNAs through direct polymerase-RBP interactions during transcription. We show that loss of TDP-43, which associates with the viral mRNAs, results in lower levels of viral mRNAs within infected cells, and a decreased yield of infectious viral particles. Overall, our results uncover an important role for TDP-43 in the influenza A virus replication cycle via a direct interaction with viral mRNAs, and point to a role of the viral polymerase in orchestrating the assembly of viral mRNPs.

Published
2023

24. Queuosine modification of tRNA-Tyrosine elicits translational reprogramming and enhances growth of Vibrio cholerae with aminoglycosides

Author

Louna Fruchard, Anamaria Babosan, Andre Carvalho, Manon Lang, Blaise Li, Magalie Duchateau, Quentin Giai-Gianetto, Mariette Matondo, Frédéric Bonhomme, Céline Fabret, Olivier Namy, Valérie de Crécy-Lagard, Didier Mazel, Zeynep Baharoglu, Plasticité du Génome Bactérien - Bacterial Genome Plasticity (PGB), Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS)-Université Paris Cité (UPCité), Hub Bioinformatique et Biostatistique - Bioinformatics and Biostatistics HUB, Institut Pasteur [Paris] (IP)-Université Paris Cité (UPCité), Plateforme de Protéomique / Proteomics platform, Université Paris Cité (UPCité)-Spectrométrie de Masse pour la Biologie – Mass Spectrometry for Biology (UTechS MSBio), Institut Pasteur [Paris] (IP)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS)-Université Paris Cité (UPCité)-Institut Pasteur [Paris] (IP)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS), Chimie biologique épigénétique - Epigenetic Chemical Biology (EpiCBio), Institut de Biologie Intégrative de la Cellule (I2BC), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS), Department of Microbiology and Cell Science, University of Florida [Gainesville] (UF), University of Florida Genetics Institute (UFGI), This research was funded by the Institut Pasteur, the Centre National de la Recherche Scientifique (CNRS-UMR 3525), ANR ModRNAntibio (ANR-21-CE35-0012), ANR-LabEx [ANR-10-LABX-62-IBEID], the Fondation pour la Recherche Médicale (FRM EQU202103012569), the Institut Pasteur grant PTR 245-19 and by the National Institute of General Medical Sciences (NIGMS) grant GM70641 to V dC-L. AB was funded by Institut Pasteur Roux-Cantarini fellowship. The authors acknowledge a DIM1Health 2019 grant from the Région Ile de France to the project EpiK for the LCMS equipment., ANR-21-CE35-0012,ModRNAntibio,Adapatation phénotypique aux antibiotiques: modifications de t/rRNA(2021), and ANR-10-LABX-0062,IBEID,Integrative Biology of Emerging Infectious Diseases(2010)

Subjects
[SDV]Life Sciences [q-bio]
Abstract

Tgt is the enzyme modifying the guanine (G) in tRNAs with GUN anti-codon to queuosine (Q). tgt is required for optimal growth of Vibrio cholerae in the presence of sub-lethal aminoglycoside concentrations. We further explored here the role of the Q in the efficiency of codon decoding upon tobramycin exposure. We characterized its impact on the overall bacterial proteome, and elucidated the molecular mechanisms underlying the effects of Q modification in antibiotic translational stress response. Using molecular reporters, we showed that Q impacts the efficiency of decoding at tyrosine TAT and TAC codons. Proteomics analyses revealed that the anti-SoxR factor RsxA is better translated in the absence of tgt. RsxA displays a codon bias towards tyrosine TAT and its overabundance leads to decreased expression of genes belonging to SoxR oxidative stress regulon. We also identified conditions that regulate tgt expression. We propose that regulation of Q modification in response to environmental cues leads to translational reprogramming of genes bearing a biased tyrosine codon usage. In silico analysis further identified candidate genes possibly subject to such translational regulation, among which DNA repair factors. Such transcripts, fitting the definition of modification tunable transcripts, are plausibly central in the bacterial response to antibiotics.

Published
2023

25. Interaction network among de novo purine nucleotide biosynthesis enzymes in Escherichia coli

Author

Antoine Gedeon, Gouzel Karimova, Nour Ayoub, Julien Dairou, Quentin Giai Gianetto, Sophie Vichier‐Guerre, Pierre‐Olivier Vidalain, Daniel Ladant, Hélène Munier‐Lehmann, Chimie et Biocatalyse, Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS), Université Paris Cité (UPCité), Biochimie des Interactions Macromoléculaires / Biochemistry of Macromolecular Interactions, Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS)-Université Paris Cité (UPCité), Laboratoire de Chimie et de Biochimie Pharmacologiques et Toxicologiques (LCBPT - UMR 8601), Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS)-Université Paris Cité (UPCité), Hub Bioinformatique et Biostatistique - Bioinformatics and Biostatistics HUB, Institut Pasteur [Paris] (IP)-Université Paris Cité (UPCité), Plateforme de Protéomique / Proteomics platform, Université Paris Cité (UPCité)-Spectrométrie de Masse pour la Biologie – Mass Spectrometry for Biology (UTechS MSBio), Institut Pasteur [Paris] (IP)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS)-Université Paris Cité (UPCité)-Institut Pasteur [Paris] (IP)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS), Spectrométrie de Masse pour la Biologie – Mass Spectrometry for Biology (UTechS MSBio), Institut Pasteur [Paris] (IP)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS)-Université Paris Cité (UPCité), Chimie biologique épigénétique - Epigenetic Chemical Biology (EpiCBio), Centre International de Recherche en Infectiologie (CIRI), École normale supérieure de Lyon (ENS de Lyon)-Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Université Jean Monnet - Saint-Étienne (UJM)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), and This work was supported in part by the Centre National de la Recherche Scientifique (CNRS), the Institut National de la Santé Et de la Recherche Médicale (INSERM), and the Institut Pasteur.Antoine Gedeon and Nour Ayoub acknowledge PhD fellowships from the Médicament, Toxicologie, Chimieet Imageries PhD school (MTCI, ED 563), Université Paris Cité.

Subjects
bacterial two-hybrid system, [SDV]Life Sciences [q-bio], Escherichia coli, Cell Biology, de novo purine biosynthesis, Molecular Biology, Biochemistry, supramolecular assembly
Abstract

International audience; In human cells, de novo purine nucleotide biosynthesis is known to be regulated through the formation of a metabolon called purinosome. Here, we employed a bacterial two-hybrid approach to characterize the protein-protein interactions network among the corresponding enzymes of Escherichia coli. Our study revealed a dense network of binary interactions that connect most purine nucleotide biosynthesis enzymes. Notably, PurK, an exclusive prokaryotic enzyme, appears as one of the central hubs of this network. We further showed that modifications in PurK, which disrupted several interactions in the network, affected the purine nucleotide pools and altered the bacterial fitness. Our data suggest that the bacterial de novo purine nucleotide biosynthesis enzymes can assemble in a supramolecular complex and that proper interactions among the components of this complex can contribute to bacterial fitness.

Published
2023
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28. In-Depth characterization of the clostridioides difficile phosphoproteome to identify Ser/Thr kinase substrates

Author

Transito Garcia-Garcia, Thibaut Douché, Quentin Giai Gianetto, Sandrine Poncet, Nesrine El Omrani, Wiep Klaas Smits, Elodie Cuenot, Mariette Matondo, Isabelle Martin-Verstraete, Pathogénèse des Bactéries Anaérobies / Pathogenesis of Bacterial Anaerobes (PBA (U-Pasteur_6)), Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS)-Université Paris Cité (UPCité), Plateforme de Protéomique / Proteomics platform, Université Paris Cité (UPCité)-Spectrométrie de Masse pour la Biologie – Mass Spectrometry for Biology (UTechS MSBio), Institut Pasteur [Paris] (IP)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS)-Université Paris Cité (UPCité)-Institut Pasteur [Paris] (IP)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS), Hub Bioinformatique et Biostatistique - Bioinformatics and Biostatistics HUB, Institut Pasteur [Paris] (IP)-Université Paris Cité (UPCité), MICrobiologie de l'ALImentation au Service de la Santé (MICALIS), AgroParisTech-Université Paris-Saclay-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE), Department of Medical Microbiology, Leiden University Medical Center (LUMC), Universiteit Leiden-Universiteit Leiden, Universiteit Leiden, Institut Universitaire de France (IUF), Ministère de l'Education nationale, de l’Enseignement supérieur et de la Recherche (M.E.N.E.S.R.), This work was funded by the Institut Pasteur, the Université Paris Cité, and grants from the ITN Marie Curie, Clospore (grant no.: H2020-MSCA-ITN-2014 642068), and the ANR DifKin (grant no.: ANR-17-CE15-0018-01)., ANR-17-CE15-0018,DifKin,Une Ser/Thr kinase impliquée dans la résistance à des composés antimicrobiens importants pour la colonization chez Clostridium difficile(2017), and European Project: 642068,H2020,H2020-MSCA-ITN-2014,CLOSPORE(2015)

Subjects
Threonine, cell division, Proteome, envelope homeostasis, Clostridioides difficile, [SDV]Life Sciences [q-bio], Protein Serine-Threonine Kinases, Phosphoproteins, Biochemistry, serine phosphorylation, Analytical Chemistry, phosphatase, Clostridioides, Bacterial Proteins, Hanks kinase, Phosphoprotein Phosphatases, Serine, threonine phosphorylation, Phosphorylation, Molecular Biology
Abstract

International audience; Clostridioides difficile is the leading cause of postantibiotic diarrhea in adults. During infection, the bacterium must rapidly adapt to the host environment by using survival strategies. Protein phosphorylation is a reversible post-translational modification employed ubiquitously for signal transduction and cellular regulation. Hanks-type serine/threonine kinases (STKs) and serine/threonine phosphatases have emerged as important players in bacterial cell signaling and pathogenicity. C. difficile encodes two STKs (PrkC and CD2148) and one phosphatase. We optimized a titanium dioxide phosphopeptide enrichment approach to determine the phosphoproteome of C. difficile. We identified and quantified 2500 proteins representing 63% of the theoretical proteome. To identify STK and serine/threonine phosphatase targets, we then performed comparative large-scale phosphoproteomics of the WT strain and isogenic ΔprkC, CD2148, Δstp, and prkC CD2148 mutants. We detected 635 proteins containing phosphorylated peptides. We showed that PrkC is phosphorylated on multiple sites in vivo and autophosphorylates in vitro. We were unable to detect a phosphorylation for CD2148 in vivo, whereas this kinase was phosphorylated in vitro only in the presence of PrkC. Forty-one phosphoproteins were identified as phosphorylated under the control of CD2148, whereas 114 proteins were phosphorylated under the control of PrkC including 27 phosphoproteins more phosphorylated in the ∆stp mutant. We also observed enrichment for phosphothreonine among the phosphopeptides more phosphorylated in the Δstp mutant. Both kinases targeted pathways required for metabolism, translation, and stress response, whereas cell division and peptidoglycan metabolism were more specifically controlled by PrkC-dependent phosphorylation in agreement with the phenotypes of the ΔprkC mutant. Using a combination of approaches, we confirmed that FtsK was phosphorylated in vivo under the control of PrkC and that Spo0A was a substrate of PrkC in vitro. This study provides a detailed mapping of kinase-substrate relationships in C. difficile, paving the way for the identification of new biomarkers and therapeutic targets.

Published
2022
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29. Statistical Analysis of Post-Translational Modifications Quantified by Label-Free Proteomics Across Multiple Biological Conditions with R: Illustration from SARS-CoV-2 Infected Cells

Author

Quentin, Giai Gianetto

Subjects
Proteomics, SARS-CoV-2, Humans, COVID-19, Peptides, Protein Processing, Post-Translational, Software
Abstract

Protein post-translational modifications (PTMs) are essential elements of cellular communication. Their variations in abundance can affect cellular pathways, leading to cellular disorders and diseases. A widely used method for revealing PTM-mediated regulatory networks is their label-free quantitation (LFQ) by high-resolution mass spectrometry. The raw data resulting from such experiments are generally interpreted using specific software, such as MaxQuant, MassChroQ, or Proline for instance. They provide data matrices containing quantified intensities for each modified peptide identified. Statistical analyses are then necessary (1) to ensure that the quantified data are of good enough quality and sufficiently reproducible, (2) to highlight the modified peptides that are differentially abundant between the biological conditions under study. The objective of this chapter is therefore to provide a complete data analysis pipeline for analyzing the quantified values of modified peptides in presence of two or more biological conditions using the R software. We illustrate our pipeline starting from MaxQuant outputs dealing with the analysis of A549-ACE2 cells infected by SARS-CoV-2 at different time stamps, freely available on PRIDE (PXD020019).

Published
2022

30. A balance between actin and Eps8/IRSp53 utilization in branched versus linear actin networks determines tunneling nanotube formation

Author

J. Michael Henderson, Nina Ljubojevic, Thibault Chaze, Daryl Castaneda, Aude Battistella, Quentin Giai Gianetto, Mariette Matondo, Stéphanie Descroix, Patricia Bassereau, and Chiara Zurzolo

Abstract

Tunneling nanotubes (TNTs) connect distant cells and mediate cargo transfer for intercellular communication in physiological and pathological contexts. How cells generate these actin-mediated protrusions to span lengths beyond those attainable by canonical filopodia remains unknown. Through a combination of micropatterning, microscopy and optical tweezer-based approaches, we demonstrate that TNTs forming through the outward extension of actin (not through cellular dislodgement) achieve distances greater than the mean length of filopodia, and that branched Arp2/3-dependent pathways attenuate the extent to which actin polymerizes in nanotubes, limiting TNT occurrence. Proteomic analysis using Epidermal growth factor receptor kinase substrate 8 (Eps8) as a positive effector of TNTs showed that upon Arp2/3 inhibition, proteins enhancing filament turnover and depolymerization were reduced and Eps8 instead exhibited heightened interactions with the inverted Bin/Amphiphysin/Rvs (I-BAR) domain protein IRSp53 that provides a direct connection with linear actin polymerases. Our data reveals how common protrusion players (Eps8 and IRSp53) form TNTs, and that when competing pathways overutilizing such proteins and monomeric actin in Arp2/3 networks are inhibited, processes promoting linear actin growth dominate to favour TNT formation. Thus, this work reinforces a general principle for actin network control for cellular protrusions where simple shifts in the balance between processes that inhibit actin growth versus those that promote growth dictate protrusion formation and the ultimate length scales protrusions achieve.

Published
2022
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31. The Flemmingsome reveals an ESCRT-to-membrane coupling via ALIX/syntenin/syndecan-4 required for completion of cytokinesis

Author

Quentin Giai Gianetto, Neetu Gupta-Rossi, Mariette Matondo, Stéphane Frémont, Frédérique Cuvelier, Audrey Salles, Julia Chamot-Rooke, Hervé Ménager, Florine Milin, Sandrine Schmutz, Cyril Addi, Murielle Rocancourt, Magalie Duchateau, Arnaud Echard, Thibaut Douché, Adrien Presle, Pascale Zimmermann, Trafic membranaire et Division cellulaire - Membrane Traffic and Cell Division, Institut Pasteur [Paris]-Centre National de la Recherche Scientifique (CNRS), Collège doctoral [Sorbonne universités], Sorbonne Université (SU), Cytometrie et Biomarqueurs – Cytometry and Biomarkers (UTechS CB), Institut Pasteur [Paris], Spectrométrie de Masse pour la Biologie – Mass Spectrometry for Biology (UTechS MSBio), Institut Pasteur [Paris]-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS), Plateforme de Protéomique / Proteomics platform, Institut Pasteur [Paris]-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS)-Institut Pasteur [Paris]-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS), Hub Bioinformatique et Biostatistique - Bioinformatics and Biostatistics HUB, BioImagerie Photonique – Photonic BioImaging (UTechS PBI), Centre de Recherche en Cancérologie de Marseille (CRCM), Centre National de la Recherche Scientifique (CNRS)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Institut Paoli-Calmettes, Fédération nationale des Centres de lutte contre le Cancer (FNCLCC)-Fédération nationale des Centres de lutte contre le Cancer (FNCLCC)-Aix Marseille Université (AMU), Catholic University of Leuven - Katholieke Universiteit Leuven (KU Leuven), This work has been supported by Institut Pasteur, CNRS, and ANR (AbCyStem, Cytosign) to A.E. C.A. received a fellowship from the Doctoral School Complexité du Vivant ED515, contrat n° 2412/2016 and AMX. A.P. received a fellowship from the Doctoral School Complexité du Vivant ED515, contrat n°2611 bis/2016 and Fondation ARC pour la recherche sur le cancer (DOC20190508876)., We thank R. Basto, G. Hickson, J. Mathieu, J.-R. Huyhn, R. Shaughnessy, M. Serres, and T. Wai for critical reading of the paper, the Echard Lab members for helpful discussions, the Recombinant antibodies platform (TAb-IP, Institut Curie, Paris) and the DSHB (University of Iowa) for antibodies. GFP-MKLP2 cells were from the Hyman Lab MPI-MCBG Dresden45., UTechS PBI is part of the France–BioImaging infrastructure network (FBI) supported by the French National Research Agency (ANR-10-INSB-04, Investments for the Future), and acknowledges support from ANR/FBI and the Région Ile-de-France (program 'Domaine d’Intérêt Majeur-Malinf') for the use of the Zeiss LSM 780 Elyra PS1 microscope. We thank P.H. Commere from the Utechs CB, Institut Pasteur for FACS sorting., Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS), Collège Doctoral, Institut Pasteur [Paris] (IP), Institut Pasteur [Paris] (IP)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS), Institut Pasteur [Paris] (IP)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS)-Institut Pasteur [Paris] (IP)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS), Aix Marseille Université (AMU)-Institut Paoli-Calmettes, Fédération nationale des Centres de lutte contre le Cancer (FNCLCC)-Fédération nationale des Centres de lutte contre le Cancer (FNCLCC)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), and Salles, Audrey

Subjects
0301 basic medicine, Syntenins, SYNDECAN, [SDV]Life Sciences [q-bio], General Physics and Astronomy, Cell Cycle Proteins, Plasma protein binding, ABSCISSION, 0302 clinical medicine, ALIX, SYNTENIN, lcsh:Science, Multidisciplinary, Chemistry, MOLECULAR-MECHANISMS, Transmembrane protein, 3. Good health, Cell biology, [SDV] Life Sciences [q-bio], Multidisciplinary Sciences, Midbody, Science & Technology - Other Topics, MACHINERY, LATE STEPS, Protein Binding, Cell division, Science, BIOGENESIS, SCISSION, HIV Budding, Endosomes, macromolecular substances, General Biochemistry, Genetics and Molecular Biology, ESCRT, Article, 03 medical and health sciences, Abscission, Humans, Cytokinesis, Organelles, MIDBODY, Science & Technology, Endosomal Sorting Complexes Required for Transport, Calcium-Binding Proteins, Cell Membrane, General Chemistry, 030104 developmental biology, Membrane protein, Syndecan-4, lcsh:Q, 030217 neurology & neurosurgery, HeLa Cells
Abstract

Cytokinesis requires the constriction of ESCRT-III filaments on the side of the midbody, where abscission occurs. After ESCRT recruitment at the midbody, it is not known how the ESCRT-III machinery localizes to the abscission site. To reveal actors involved in abscission, we obtained the proteome of intact, post-abscission midbodies (Flemmingsome) and identified 489 proteins enriched in this organelle. Among these proteins, we further characterized a plasma membrane-to-ESCRT module composed of the transmembrane proteoglycan syndecan-4, ALIX and syntenin, a protein that bridges ESCRT-III/ALIX to syndecans. The three proteins are highly recruited first at the midbody then at the abscission site, and their depletion delays abscission. Mechanistically, direct interactions between ALIX, syntenin and syndecan-4 are essential for proper enrichment of the ESCRT-III machinery at the abscission site, but not at the midbody. We propose that the ESCRT-III machinery must be physically coupled to a membrane protein at the cytokinetic abscission site for efficient scission, uncovering common requirements in cytokinesis, exosome formation and HIV budding., ESCRT filaments drive the final abscission between two daughter cells but how they physically interact with the membrane is unclear. Using proteomics, the authors show that syndecan-4/syntenin/ALIX couples the ESCRT machinery to the abscission site and thus promotes efficient abscission.

Published
2020
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32. Single locus phosphoproteomics reveals phosphorylation of RPA-1 is required for generation of single-strand DNA following a break at a subtelomeric locus

Author

Emilia McLaughlin, Annick Dujeancourt-Henry, Thibault Chaze, Quentin Giai Gianetto, Mariette Matondo, Michael D Urbaniak, Lucy Glover, Biologie moléculaire des Trypanosomes - Trypanosome Molecular Biology, Institut Pasteur [Paris] (IP)-Université Paris Cité (UPCité), Plateforme de Protéomique / Proteomics platform, Université Paris Cité (UPCité)-Spectrométrie de Masse pour la Biologie – Mass Spectrometry for Biology (UTechS MSBio), Institut Pasteur [Paris] (IP)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS)-Université Paris Cité (UPCité)-Institut Pasteur [Paris] (IP)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS), Hub Bioinformatique et Biostatistique - Bioinformatics and Biostatistics HUB, Division of Biomedical and Life Sciences, Faculty of Health and Medicine, Lancaster University, Work in the LG laboratory is has received financial support from the Institut Pasteur (G5 Junior group). EJM is part of the Pasteur - Paris University (PPU) International PhD Program. This project has received funding from the European Union's Horizon 2020 research and innovation programme under the Marie Sklodowska-Curie grant agreement No 665807 and from the Foundation Recherché Médicale grant number FDT202012010602. Funding for open access charge: Institut Pasteur G5 funding., EJM, MM, MU and LG designed the experiments. EJM, ADH, TC performed the experiments,EJM, QGG and MU performed the statistical analysis. EJM and LG wrote the manuscript, all authors edited the manuscript. We would also like to thank Sebastian Hutchinson for his guidance with the analysis of the VSG-sequencing, and European Project: 665807,H2020,H2020-MSCA-COFUND-2014,PASTEURDOC(2015)

Subjects
Phosphoproteomics, [SDV]Life Sciences [q-bio], DNA damage, Trypanosoma brucei
Abstract

Damage to the genetic material of the cell poses a universal threat to all forms of life. Central to the DNA damage response (DDR) is a phosphorylation signalling cascade that leads to the co-ordination of the cellular response to a DNA break. Identifying the proteins that are phosphorylated is crucial to understanding the mechanisms underlying this DDR. We have used SILAC-based quantitative phosphoproteomics to profile changes in phosphorylation site abundance following a single double strand break (DSB) at a chromosome internal locus and the subtelomeric bloodstream form expression site in Trypanosoma brucei. We report >6500 phosphorylation sites, including a core set of 211 DSB responsive phosphorylation sites. Along with phosphorylation of canonical DNA damage factors, we find that there is a striking distinction between the proteins phosphorylated in response to a chromosome internal DSB and one at the active BES and describe a single phosphorylation event on Replication factor A (RPA) 1 that is required for efficient resection at a bloodstream form expression site.

Published
2022
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33. Biological differences between FIM2 and FIM3 fimbriae of Bordetella pertussis: not just the serotype

Author

Soraya Matczak, Valérie Bouchez, Pauline Leroux, Thibaut Douché, Nils Collinet, Annie Landier, Quentin Giai Gianetto, Sophie Guillot, Julia Chamot-Rooke, Milena Hasan, Mariette Matondo, Sylvain Brisse, Julie Toubiana, Biodiversité et Epidémiologie des Bactéries pathogènes - Biodiversity and Epidemiology of Bacterial Pathogens, Institut Pasteur [Paris] (IP)-Université Paris Cité (UPCité), Plateforme de Protéomique / Proteomics platform, Université Paris Cité (UPCité)-Spectrométrie de Masse pour la Biologie – Mass Spectrometry for Biology (UTechS MSBio), Institut Pasteur [Paris] (IP)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS)-Université Paris Cité (UPCité)-Institut Pasteur [Paris] (IP)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS), Centre national de Référence de la Coqueluche et autres Bordetelloses - National Reference Center for Whooping Cough and other Bordetella infections (CNR), Hub Bioinformatique et Biostatistique - Bioinformatics and Biostatistics HUB, Spectrométrie de Masse pour la Biologie – Mass Spectrometry for Biology (UTechS MSBio), Institut Pasteur [Paris] (IP)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS)-Université Paris Cité (UPCité), Cytometrie et Biomarqueurs – Cytometry and Biomarkers (UTechS CB), Département de Pédiatrie et maladies infectieuses [CHU Necker], CHU Necker - Enfants Malades [AP-HP], Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP), JT received funding from Ville de Paris as part of the EMERGENCE program. SM received funding from la Fondation pour la Recherche Médicale (FRM). SB received funding from INCEPTION program (ANR-16-CONV-0005)., and ANR-16-CONV-0005,INCEPTION,Institut Convergences pour l'étude de l'Emergence des Pathologies au Travers des Individus et des populatiONs(2016)

Subjects
Infectious Diseases, [SDV]Life Sciences [q-bio], Immunology, Microbiology, Microbes and Infection
Abstract

International audience; Introduction: Bordetella pertussis still circulates worldwide despite vaccination. Fimbriae are components of some acellular pertussis vaccines. Population fluctuations of B. pertussis fimbrial serotypes (FIM2 and FIM3) are observed, and fim3 alleles (fim3-1 [clade 1] and fim3-2 [clade 2]) mark a major phylogenetic subdivision of B. pertussis. Objectives: To compare microbiological characteristics and expressed protein profiles between fimbrial serotypes FIM2 and FIM3 and genomic clades. Methods: A total of 23 isolates were selected. Absolute protein abundance of the main virulence factors, autoagglutination and biofilm formation, bacterial survival in whole blood, induced blood cell cytokine secretion, and global proteome profiles were assessed. Results: Compared to FIM3, FIM2 isolates produced more fimbriae, less cellular pertussis toxin subunit 1 and more biofilm, but auto-agglutinated less. FIM2 isolates had a lower survival rate in cord blood, but induced higher levels of IL-4, IL-8 and IL-1 secretion. Global proteomecomparisons uncovered 15 differentially produced proteins between FIM2 and FIM3 isolates, involved in adhesion and metabolism of metals. FIM3 isolates of clade 2 produced more FIM3 and more biofilm compared to clade 1. Conclusion: FIM serotype and fim3 clades are associated with proteomic and other biological differences, which may have implications on pathogenesis and epidemiological emergence.

Published
2023
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36. Purification of infection-associated macropinosomes by magnetic isolation for proteomic characterization

Author

Quentin Giai Gianetto, Magalie Duchateau, Virginie Stévenin, Mariette Matondo, Jost Enninga, Yuen-Yan Chang, Dynamique des Interactions Hôte-Pathogène - Dynamics of Host-Pathogen Interactions, Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS)-Université Paris Cité (UPCité), Leiden University Medical Center (LUMC), Universiteit Leiden, Plateforme de Protéomique / Proteomics platform, Université Paris Cité (UPCité)-Spectrométrie de Masse pour la Biologie – Mass Spectrometry for Biology (UTechS MSBio), Institut Pasteur [Paris] (IP)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS)-Université Paris Cité (UPCité)-Institut Pasteur [Paris] (IP)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS), Hub Bioinformatique et Biostatistique - Bioinformatics and Biostatistics HUB, Institut Pasteur [Paris] (IP)-Université Paris Cité (UPCité), National Institutes of Health [Bethesda] (NIH), Y-Y.C was supported by a fellowship from the Fondation pour la Recherche Médicale (FRM, SPF20160936275). V.S. was supported by a PhD fellowship from the University Paris Diderot (now Université de Paris) allocated by the ENS Paris-Saclay, a grant from the FRM (FRM, FDT20170436843), and an extension grant from the BCI Department of Institut Pasteur. J.E. is a member of the LabEx consortia IBEID and MilieuInterieur. J.E. also acknowledges support from the ANR (grant StopBugEntry and AutoHostPath) and the ERC (CoG EndoSubvert)., ANR-10-LABX-0062,IBEID,Integrative Biology of Emerging Infectious Diseases(2010), ANR-10-LABX-0069,MILIEU INTERIEUR,GENETIC & ENVIRONMENTAL CONTROL OF IMMUNE PHENOTYPE VARIANCE: ESTABLISHING A PATH TOWARDS PERSONALIZED MEDICINE(2010), ANR-15-CE15-0017,StopBugEntry,Identification des nouvelles molécules cellulaires cibles pour combattre les infections bactériennes(2015), ANR-15-CE15-0018,AutoHostPath,Rôles alternatifs pour les récepteurs de l'autophagie dans les interactions hôte-pathogène(2015), European Project: 682809,EndoSubvert(2017), Institut Pasteur [Paris]-Centre National de la Recherche Scientifique (CNRS), Spectrométrie de Masse pour la Biologie – Mass Spectrometry for Biology (UTechS MSBio), and Institut Pasteur [Paris]-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS)-Institut Pasteur [Paris]-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS)

Subjects
Proteomics, 0303 health sciences, Chemistry, Vesicle, Pinocytosis, Production cost, Magnetic Phenomena, Endocytic cycle, Cell migration, Composition analysis, General Biochemistry, Genetics and Molecular Biology, 3. Good health, Cell biology, 03 medical and health sciences, 0302 clinical medicine, Endocytic vesicle, Eukaryotic Cells, Magnetic isolation, Cell Movement, [SDV.BC.IC]Life Sciences [q-bio]/Cellular Biology/Cell Behavior [q-bio.CB], [SDV.BBM.BC]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biochemistry [q-bio.BM], 030217 neurology & neurosurgery, 030304 developmental biology
Abstract

International audience; Macropinocytosis refers to the nonselective uptake of extracellular molecules into many different types of eukaryotic cells within large fluid-filled vesicles named macropinosomes. Macropinosomes are relevant for a wide variety of cellular processes, such as antigen sampling in immune cells, homeostasis in the kidney, cell migration or pathogen uptake. Understanding the molecular composition of the different macropinosomes formed during these processes has helped to differentiate their regulations from other endocytic events. Here, we present a magnetic purification protocol that segregates scarce macropinosomes from other endocytic vesicles at a high purity and in a low-cost and unbiased manner. Our protocol takes advantage of moderate-sized magnetic beads of 100 nm in diameter coupled to mass-spectrometry-based proteomic analysis. Passing the cell lysate through a table-top magnet allows the quick retention of the bead-containing macropinosomes. Unlike other cell-fractionation-based methodologies, our protocol minimizes sample loss and production cost without prerequisite knowledge of the macropinosomes and with minimal laboratory experience. We describe a detailed procedure for the isolation of infection-associated macropinosomes during bacterial invasion and the optimization steps to readily adapt it to various studies. The protocol can be performed in 3 d to provide highly purified and enriched macropinosomes for qualitative proteomic composition analysis.

Published
2021
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37. Mitochondrial fission process 1 (MTFP1) controls bioenergetic efficiency and prevents inflammatory cardiomyopathy and heart failure in mice

Author

Timothy Wai, Erminia Donnarumma, Christoph Maack, Thibault Chaze, Quentin Giai Gianetto, Mariette Matondo, Elodie Vimont, Etienne Kornobis, Michael Kohlhaas, Maryse Moya-Nilges, Biologie mitochondriale – Mitochondrial biology, Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS)-Université Paris Cité (UPCité), University Clinic Würzburg, Biomics (plateforme technologique), Institut Pasteur [Paris] (IP)-Université Paris Cité (UPCité), Hub Bioinformatique et Biostatistique - Bioinformatics and Biostatistics HUB, Plateforme de Spectrométrie de Masse Protéomique - Mass Spectrometry Proteomics Platform (MSPP), and Centre National de la Recherche Scientifique (CNRS)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE)-Université de Montpellier (UM)

Subjects
uncoupling, Programmed cell death, permeability transition pore, Cardiac fibrosis, Chemistry, oxidative phosphorylation, Cardiomyopathy, Oxidative phosphorylation, Mitochondrion, medicine.disease, Cell biology, mitochondria, Mitochondrial permeability transition pore, Knockout mouse, medicine, [SDV.SPEE]Life Sciences [q-bio]/Santé publique et épidémiologie, Mitochondrial fission, cardiomyopathy
Abstract

Mitochondria are paramount to the metabolism and survival of cardiomyocytes. Here we show that Mitochondrial Fission Process 1 (MTFP1) is essential for cardiac structure and function. Constitutive knockout of cardiomyocyte MTFP1 in mice resulted in adult-onset dilated cardiomyopathy (DCM) characterized by sterile inflammation and cardiac fibrosis that progressed to heart failure and middle-aged death. Failing hearts from cardiomyocyte-restricted knockout mice displayed a general decline in mitochondrial gene expression and oxidative phosphorylation (OXPHOS) activity. Pre-DCM, we observed no defects in mitochondrial morphology, content, gene expression, OXPHOS assembly nor phosphorylation dependent respiration. However, knockout cardiac mitochondria displayed reduced membrane potential and increased non-phosphorylation dependent respiration, which could be rescued by pharmacological inhibition of the adenine nucleotide translocase ANT. Primary cardiomyocytes from pre-symptomatic knockout mice exhibited normal excitation-contraction coupling but increased sensitivity to programmed cell death (PCD), which was accompanied by an opening of the mitochondrial permeability transition pore (mPTP). Intriguingly, mouse embryonic fibroblasts deleted for Mtfp1 recapitulated PCD sensitivity and mPTP opening, both of which could be rescued by pharmacological or genetic inhibition of the mPTP regulator Cyclophilin D. Collectively, our data demonstrate that contrary to previous in vitro studies, the loss of the MTFP1 promotes mitochondrial uncoupling and increases cell death sensitivity, causally mediating pathogenic cardiac remodeling.

Published
2021
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38. Listeriolysin S: A bacteriocin from

Author

Jazmín, Meza-Torres, Mickaël, Lelek, Juan J, Quereda, Martin, Sachse, Giulia, Manina, Dmitry, Ershov, Jean-Yves, Tinevez, Lilliana, Radoshevich, Claire, Maudet, Thibault, Chaze, Quentin, Giai Gianetto, Mariette, Matondo, Marc, Lecuit, Isabelle, Martin-Verstraete, Christophe, Zimmer, Hélène, Bierne, Olivier, Dussurget, Pascale, Cossart, and Javier, Pizarro-Cerdá

Subjects
Cytoplasm, Cell Membrane, Biological Sciences, microfluidic microscopy, Listeria monocytogenes, Microbiology, contact-dependent inhibition (CDI), Hemolysin Proteins, Adenosine Triphosphate, Bacteriocins, bacteriocin, Listeria monocytogenes (Lm), Listeriolysin S (LLS)
Abstract

Significance Listeria monocytogenes (Lm) is a bacterial pathogen that causes listeriosis, a foodborne disease characterized by gastroenteritis, meningitis, bacteremia, and abortions in pregnant women. The most severe human listeriosis outbreaks are associated with a subset of Lm hypervirulent clones that encode the bacteriocin Listeriolysin S (LLS), which modifies the gut microbiota and allows efficient Lm gut colonization and invasion of deeper organs. Our present work identifies the killing mechanism displayed by LLS to outcompete gut commensal bacteria, demonstrating that it induces membrane permeabilization and membrane depolarization of target bacteria. Moreover, we show that LLS is a thiazole/oxazole–modified microcin that displays a contact-dependent inhibition mechanism., Listeriolysin S (LLS) is a thiazole/oxazole–modified microcin (TOMM) produced by hypervirulent clones of Listeria monocytogenes. LLS targets specific gram-positive bacteria and modulates the host intestinal microbiota composition. To characterize the mechanism of LLS transfer to target bacteria and its bactericidal function, we first investigated its subcellular distribution in LLS-producer bacteria. Using subcellular fractionation assays, transmission electron microscopy, and single-molecule superresolution microscopy, we identified that LLS remains associated with the bacterial cell membrane and cytoplasm and is not secreted to the bacterial extracellular space. Only living LLS-producer bacteria (and not purified LLS-positive bacterial membranes) display bactericidal activity. Applying transwell coculture systems and microfluidic-coupled microscopy, we determined that LLS requires direct contact between LLS-producer and -target bacteria in order to display bactericidal activity, and thus behaves as a contact-dependent bacteriocin. Contact-dependent exposure to LLS leads to permeabilization/depolarization of the target bacterial cell membrane and adenosine triphosphate (ATP) release. Additionally, we show that lipoteichoic acids (LTAs) can interact with LLS and that LTA decorations influence bacterial susceptibility to LLS. Overall, our results suggest that LLS is a TOMM that displays a contact-dependent inhibition mechanism.

Published
2021

40. Amphiregulin mediates non-cell-autonomous effect of senescence on reprogramming

Author

Mathieu von Joest, Cheng Chen, Thibaut Douché, Jeremy Chantrel, Aurélie Chiche, Quentin Giai Gianetto, Mariette Matondo, Han Li, Plasticité cellulaire et Modélisation des Maladies / Cellular Plasticity and Disease Modelling, Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS)-Université Paris Cité (UPCité), Plateforme de Protéomique / Proteomics platform, Université Paris Cité (UPCité)-Spectrométrie de Masse pour la Biologie – Mass Spectrometry for Biology (UTechS MSBio), Institut Pasteur [Paris] (IP)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS)-Université Paris Cité (UPCité)-Institut Pasteur [Paris] (IP)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS), Hub Bioinformatique et Biostatistique - Bioinformatics and Biostatistics HUB, Institut Pasteur [Paris] (IP)-Université Paris Cité (UPCité), Work in the laboratory of H.L. is funded by Institut Pasteur, Centre National pour la Recherche Scientific, and the Agence Nationale de la Recherche, France (Laboratoire d’Excellence Revive, Investissement d’Avenir, ANR-10-LABX-73, ANR-16-CE13-0017, ANR-21-CE13-0006-01). A.C. was funded by the postdoctoral fellowships from the Revive Consortium., We are indebted to Jun Zhang for his excellent technical support in image analysis. We are grateful to the Central Animal Facility, the Proteomic Platform, the Cytometry Platform, and the Bioinformatics and Biostatistics Hub of the Institut Pasteur., ANR-10-LABX-0073,REVIVE,Stem Cells in Regenerative Biology and Medicine(2010), ANR-16-CE13-0017,iCELLPLASTICITY,Plasticité cellulaire dans le vieillissement(2016), and ANR-21-CE13-0006,SenoMuscle,Conséquence de la sénescence dans la régénération musculaire et les myopathies dégénératives(2021)

Subjects
muscle regeneration, quantitative proteomics, EGFR, [SDV]Life Sciences [q-bio], aging, Cell Plasticity, cellular plasticity, SASP, Amphiregulin, General Biochemistry, Genetics and Molecular Biology, in vitro reprogramming, Phenotype, in vivo reprogramming, CP: Cell biology, Cellular Senescence, Signal Transduction
Abstract

International audience; Cellular senescence is an irreversible growth arrest with a dynamic secretome, termed the senescence-associatedsecretory phenotype (SASP). Senescence is a cell-intrinsic barrier for reprogramming, whereas theSASP facilitates cell fate conversion in non-senescent cells. However, the mechanisms by which reprogramming-induced senescence regulates cell plasticity are not well understood. Here, we investigate how the heterogeneityof paracrine senescence impacts reprogramming. We show that senescence promotes in vitro reprogrammingin a stress-dependent manner. Unbiased proteomics identifies a catalog of SASP factorsinvolved in the cell fate conversion. Amphiregulin (AREG), frequently secreted by senescent cells, promotesin vitro reprogramming by accelerating proliferation and the mesenchymal-epithelial transition via EGFRsignaling. AREG treatment diminishes the negative effect of donor age on reprogramming. Finally, AREG enhances in vivo reprogramming in skeletal muscle. Hence, various SASP factors can facilitate cellular plasticityto promote reprogramming and tissue repair.

Published
2022
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41. RNase E-HupB Dynamic Interaction Fosters Mycobacterial Cell Homeostasis and Fitness

Author

Mariette Matondo, Quentin Giai Gianetto, Thibaut Douché, Giulia Manina, and Anna Griego

Subjects
History, Polymers and Plastics, Cellular adaptation, RNase P, Ribonuclease E, RNA, Cellular homeostasis, MRNA stabilization, Biology, Interactome, Industrial and Manufacturing Engineering, Cell biology, Gene expression, Business and International Management
Abstract

RNA turnover is a primary source of gene expression variation, in turn promoting cellular adaptation. Mycobacteria leverage reversible mRNA stabilization to endure hostile conditions. Although ribonuclease E is essential for RNA turnover in several species, its role in mycobacterial single-cell physiology and functional phenotypic diversification remains unexplored. Here, by integrating live-single-cell and quantitative-mass-spectrometry approaches, we show that ribonuclease E forms dynamic foci, which are associated with cellular homeostasis and single-cell fate, and we discover a versatile molecular interactome. In particular, we prove the interaction between ribonuclease E and the nucleoid-associated protein HupB, which is particularly pronounced during drug treatment and infection, where we also observed marked increase of cell-to-cell phenotypic diversity. Disruption of ribonuclease E expression affects HupB levels, impairing Mycobacterium tuberculosis growth homeostasis during treatment, intracellular replication and host spread. Our work lays the foundation for targeting the ribonuclease E and its molecular partner HupB, aiming to undermine Mycobacterium tuberculosis cellular balance, diversification capacity and persistence.

Published
2021
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42. Reducing neutrophil exposure to oxygen allows their basal state maintenance

Author

Benoit S. Marteyn, Véronique Witko-Sarsat, Louise Injarabian, Jurate Skerniskyte, Quentin Giai Gianetto, Giai Gianetto, Quentin, Effet de l'exposition des neutrophiles à l'oxygène sur leur activation et leur mort : une lame à double tranchant - - NEUTROXIA2017 - ANR-17-CE15-0012 - AAPG2017 - VALID, Architecture et Réactivité de l'ARN (ARN), Institut de biologie moléculaire et cellulaire (IBMC), Université de Strasbourg (UNISTRA)-Centre National de la Recherche Scientifique (CNRS)-Université de Strasbourg (UNISTRA)-Centre National de la Recherche Scientifique (CNRS)-Centre National de la Recherche Scientifique (CNRS), Institut de biochimie et génétique cellulaires (IBGC), Université Bordeaux Segalen - Bordeaux 2-Centre National de la Recherche Scientifique (CNRS), University Hospital of Cologne [Cologne], Hub Bioinformatique et Biostatistique - Bioinformatics and Biostatistics HUB, Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS), Plateforme de Protéomique / Proteomics platform, Spectrométrie de Masse pour la Biologie – Mass Spectrometry for Biology (UTechS MSBio), Institut Pasteur [Paris] (IP)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS)-Institut Pasteur [Paris] (IP)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS), Institut Cochin (IC UM3 (UMR 8104 / U1016)), Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Université Paris Cité (UPCité), Pathogénèse des Infections vasculaires / Pathogenesis of Vascular Infections, Institut Pasteur [Paris] (IP)-Institut National de la Santé et de la Recherche Médicale (INSERM), This work was supported by the ANR JCJC grant (ANR-17-CE15-0012) (BSM) and has benefitted from the support provided by the University for Advanced Study (USIAS) for a fellowship, within the French national program 'Investment for the futur (Idex-UNISTRA) (BSM), We thank Evangeline Bennana (sample preparation) and Morgan Le Gall (data processing) from the institut Cochin 3P5 Proteom'IC platform, ANR-17-CE15-0012,NEUTROXIA,Effet de l'exposition des neutrophiles à l'oxygène sur leur activation et leur mort : une lame à double tranchant(2017), Institut Pasteur [Paris]-Centre National de la Recherche Scientifique (CNRS), Institut Pasteur [Paris]-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS)-Institut Pasteur [Paris]-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS), Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Université de Paris (UP), and Institut Pasteur [Paris]-Institut National de la Santé et de la Recherche Médicale (INSERM)

Subjects
0301 basic medicine, [SDV.IMM] Life Sciences [q-bio]/Immunology, Short Communication, Immunology, chemistry.chemical_element, Oxygen, Extracellular Traps, Neutrophil Activation, 03 medical and health sciences, Basal (phylogenetics), chemistry.chemical_compound, Leukocyte Count, 0302 clinical medicine, neutrophils, [SDV.BBM.GTP]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Genomics [q-bio.GN], medicine, Immunology and Allergy, Glycolysis, DAPI, Hyperoxia, Innate immune system, viability, anoxia, Cell Biology, Neutrophil extracellular traps, Cell biology, Cytosol, 030104 developmental biology, chemistry, hyperoxia, [SDV.BBM.GTP] Life Sciences [q-bio]/Biochemistry, Molecular Biology/Genomics [q-bio.GN], [SDV.IMM]Life Sciences [q-bio]/Immunology, activation, medicine.symptom, 030215 immunology
Abstract

Neutrophils are the most abundant circulating white blood cells and are the central players of the innate immune response. During their lifecycle, neutrophils mainly evolve under low oxygen conditions (0.1–4% O2), to which they are well adapted. Neutrophils are atypical cells since they are highly glycolytic and susceptible to oxygen exposure, which induces their activation and death through mechanisms that remain currently elusive. Nevertheless, nearly all studies conducted on neutrophils are carried out under atmospheric oxygen (21%), corresponding to hyperoxia. Here, we investigated the impact of hyperoxia during neutrophil purification and culture on neutrophil viability, activation and cytosolic protein content. We demonstrate that neutrophil hyper‐activation (CD62L shedding) is induced during culture under hyperoxic conditions (24 h), compared with neutrophils cultured under anoxic conditions. Spontaneous neutrophil extracellular trap (NET) formation is observed when neutrophils face hyperoxia during purification or culture. In addition, we show that maintaining neutrophils in autologous plasma is the preferred strategy to maintain their basal state. Our results show that manipulating neutrophils under hyperoxic conditions leads to the loss of 57 cytosolic proteins during purification, while it does not lead to an immediate impact on neutrophil activation (CD11bhigh, CD54high, CD62Lneg) or viability (DAPI+). We identified two clusters of proteins belonging to cholesterol metabolism and to the complement and coagulation cascade pathways, which are highly susceptible to neutrophil oxygen exposure during neutrophil purification. In conclusion, protecting neutrophil from oxygen during their purification and culture is recommended to avoid activation and to prevent the alteration of cytosolic protein composition., In this study, we demonstrate that preserving neutrophils from oxygen exposure during their purification and culture is recommended to avoid their activation and to prevent formation of spontaneous neutrophil extracellular traps. Studying neutrophils in vitro under controlled oxygen levels similar to those encountered in vivo is essential for a better comprehension of their physiology.

Published
2021
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43. ProteoCombiner: integrating bottom-up with top-down proteomics data for improved proteoform assessment

Author

Julia Chamot-Rooke, Mariette Matondo, Mathieu Dupré, Diogo B. Lima, Magalie Duchateau, Martial Rey, Quentin Giai Gianetto, Spectrométrie de Masse pour la Biologie – Mass Spectrometry for Biology (UTechS MSBio), Centre National de la Recherche Scientifique (CNRS)-Centre de Ressources et de Recherche Technologique - Center for Technological Resources and Research (C2RT), Institut Pasteur [Paris]-Institut Pasteur [Paris], Hub Bioinformatique et Biostatistique - Bioinformatics and Biostatistics HUB, Institut Pasteur [Paris]-Centre National de la Recherche Scientifique (CNRS), This work has been supported by the ANR [project ANR-15-CE18-0021]. It is also part of the European Joint Programme One Health EJP from the European Union’s Horizon 2020 Research and Innovation Programme [grant agreement 773830]., ANR-15-CE18-0021,PathoTOP,Identification rapide de pathogènes bactériens par protéomique top-down en contexte clinique(2015), European Project: 773830,H2020-SFS-2017-1,MedVetKlebs(2018), Institut Pasteur [Paris]-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS), European Project: 773830,H2020-SFS-2017-1,MedVetKlebs (a component of European Joint Programme One Health EJP)(2018), Institut Pasteur [Paris] (IP)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS), Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS), and European Project: 773830,H2020-SFS-2017-1,MedVetKlebs (a component of European Joint Programme One Health)(2018)

Subjects
Statistics and Probability, Supplementary data, Proteomics, 0303 health sciences, Information retrieval, Proteome, Computer science, 030302 biochemistry & molecular biology, Top-down and bottom-up design, Top-down proteomics, Biochemistry, Computer Science Applications, 03 medical and health sciences, Computational Mathematics, Computational Theory and Mathematics, [CHIM.ANAL]Chemical Sciences/Analytical chemistry, Tandem Mass Spectrometry, [SDV.BBM.GTP]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Genomics [q-bio.GN], [SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology, [INFO.INFO-BI]Computer Science [cs]/Bioinformatics [q-bio.QM], Molecular Biology, Protein Processing, Post-Translational, Software, 030304 developmental biology
Abstract

Motivation We present a high-performance software integrating shotgun with top-down proteomic data. The tool can deal with multiple experiments and search engines. Enable rapid and easy visualization, manual validation and comparison of the identified proteoform sequences including the post-translational modification characterization. Results We demonstrate the effectiveness of our approach on a large-scale Escherichia coli dataset; ProteoCombiner unambiguously shortlisted proteoforms among those identified by the multiple search engines. Availability and implementation ProteoCombiner, a demonstration video and user tutorial are freely available at https://proteocombiner.pasteur.fr, for academic use; all data are thus available from the ProteomeXchange consortium (identifier PXD017618). Supplementary information Supplementary data are available at Bioinformatics online.

Published
2020
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44. Ser/Thr kinase-dependent phosphorylation of the peptidoglycan hydrolase CwlA controls its export and modulates cell division in Clostridioides difficile

Author

Mariette Matondo, Sandrine Poncet, Pascal Courtin, Marie-Pierre Chapot-Chartier, Elodie Cuenot, Transito Garcia-Garcia, Thibaut Douché, Quentin Giai Gianetto, Thomas Candela, Isabelle Martin-Verstraete, Bruno Dupuy, and Johann Peltier

Subjects
0303 health sciences, Cell division, 030306 microbiology, Chemistry, Cell growth, Kinase, Cell, Cell biology, Cell wall, 03 medical and health sciences, chemistry.chemical_compound, medicine.anatomical_structure, medicine, Phosphorylation, Peptidoglycan, Signal transduction, 030304 developmental biology
Abstract

Cell growth and division require a balance between synthesis and hydrolysis of the peptidoglycan (PG). Inhibition of PG synthesis or uncontrolled PG hydrolysis can be lethal for the cells, making it imperative to control peptidoglycan hydrolase (PGH) activity. The serine/threonine kinases (STKs) of the Hanks family control cell division and envelope homeostasis, but only a few kinase substrates and associated molecular mechanisms have been identified. In this work, we identified CwlA as the first STK-PrkC substrate in the human pathogen Clostridiodes difficile and showed that CwlA is an endopeptidase involved in daughter cell separation. We demonstrated that PrkC-dependent phosphorylation inhibits CwlA export, therefore controlling the hydrolytic activity in the cell wall. High level of CwlA at the cell surface led to cell elongation, whereas low level caused cell separation defects. We thus provided evidence that the STK signaling pathway regulates PGH homeostasis to precisely control PG hydrolysis during cell division.

Published
2020
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45. Proteomic Analysis Uncovers Measles Virus Protein C Interaction with p65/iASPP/p53 Protein Complex

Author

Hyeju Eun, Mariette Matondo, Thibaut Douché, Regis Grailhe, Frédéric Tangy, Anastassia V. Komarova, Yves Jacob, Quentin Giai Gianetto, Marc Santolini, István Kovács, Chantal Combredet, and Alice Meignié

Subjects
0303 health sciences, Programmed cell death, Innate immune system, 030302 biochemistry & molecular biology, Protein combining, Biology, biology.organism_classification, 3. Good health, Cell biology, Measles virus, 03 medical and health sciences, Viral replication, Interferon, Immunity, Cell culture, medicine, 030304 developmental biology, medicine.drug
Abstract

Viruses manipulate central machineries of host cells to their advantage. They prevent host cell antiviral responses to create a favorable environment for their survival and propagation. Measles virus (MV) encodes two non-structural proteins MV-V and MV-C known to counteract the host interferon response and to regulate cell death pathways. Several molecular mechanisms underlining MV-V regulation of innate immunity and cell death pathways have been proposed, whereas MV-C host protein partners are less studied. We suggest that some cellular factors that are controlled by MV-C protein during viral replication could be components of innate immunity and the cell death pathways. To determine which host factors are targeted by MV-C, we captured both direct and indirect host protein partners of MV-C protein. For this, we used a strategy based on recombinant viruses expressing tagged viral proteins followed by affinity purification and a bottom-up mass spectrometry analysis. From the list of host proteins specifically interacting with MV-C protein in different cell lines we selected the host targets that belong to immunity and cell death pathways for further validation. Direct protein partners of MV-C were determined by applying protein complementation assay (PCA) and the bioluminescence resonance energy transfer (BRET) approach. As a result, we found that MV-C protein specifically interacts with p65/iASPP/p53 protein complex that controls both cell death and innate immunity pathways.

Published
2020
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46. Experimental evolution links post-transcriptional regulation to Leishmania fitness gain

Author

Laura Piel, K. Shanmugha Rajan, Giovanni Bussotti, Hugo Varet, Rachel Legendre, Caroline Proux, Thibaut Douché, Quentin Giai-Gianetto, Thibault Chaze, Thomas Cokelaer, Barbora Vojtkova, Nadav Gordon-Bar, Tirza Doniger, Smadar Cohen-Chalamish, Praveenkumar Rengaraj, Céline Besse, Anne Boland, Jovana Sadlova, Jean-François Deleuze, Mariette Matondo, Ron Unger, Petr Volf, Shulamit Michaeli, Pascale Pescher, Gerald F. Späth, Parasitologie moléculaire et Signalisation / Molecular Parasitology and Signaling, Institut Pasteur [Paris] (IP)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Paris Cité (UPCité), The Everard and Mina Goodman Faculty of Life Sciences, Bar-Ilan University [Israël], Hub Bioinformatique et Biostatistique - Bioinformatics and Biostatistics HUB, Institut Pasteur [Paris] (IP)-Université Paris Cité (UPCité), Biomics (plateforme technologique), Plateforme de Protéomique / Proteomics platform, Université Paris Cité (UPCité)-Spectrométrie de Masse pour la Biologie – Mass Spectrometry for Biology (UTechS MSBio), Institut Pasteur [Paris] (IP)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS)-Université Paris Cité (UPCité)-Institut Pasteur [Paris] (IP)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS), Charles University [Prague] (CU), The Mina and Everard Goodman Faculty of Life Sciences, Centre National de Recherche en Génomique Humaine (CNRGH), Commissariat à l'énergie atomique et aux énergies alternatives (CEA), This work was supported by the Fondation de la Recherche Médicale contract FDT201805005619 (LP), the Agence Nationale pour la Recherche Labex ‘French Alliance for Parasitology and Health Care’ contract ANR-11-LABX-0024 (GFS, LP, PP, GB) and Labex ‘Integrative Biology of Emerging Infectious Diseases’ contract ANR-10-LABX-62-IBEID, the Campus France Franco-Israeli Programme Hubert Curien Maimonide 2018 (GFS and SM), the France Génomique National infrastructure, funded as part of the « Investissements d’Avenir » program managed by the Agence Nationale pour la Recherche contract ANR-10-INBS-09 (HV, RL, CP), the EU H2020 project LeiSHield-MATI - REP-778298-1 (GS), and the ERD Funds, project CePaViP (CZ.02.1.01/0.0/0.0/ 16_019/0000759) (BV, JS, PV)., We thank the CEA-CNRGH for its contribution to the sequencing costs and all the CEA-CNRGH staff who performed sample preparation and sequencing for their excellent technical assistance, ANR-11-LABX-0024,ParaFrap,Alliance française contre les maladies parasitaires(2011), ANR-10-LABX-0062,IBEID,Integrative Biology of Emerging Infectious Diseases(2010), ANR-10-INBS-0009,France-Génomique,Organisation et montée en puissance d'une Infrastructure Nationale de Génomique(2010), and European Project: 778298,H2020,H2020-MSCA-RISE-2017,LeiSHield-MATI(2018)

Subjects
Proteomics, Gene Expression Regulation, [SDV]Life Sciences [q-bio], Virology, Immunology, Genetics, Humans, Leishmaniasis, Visceral, Parasitology, Molecular Biology, Microbiology, Genomic Instability, Leishmania donovani
Abstract

The protozoan parasite Leishmania donovani causes fatal human visceral leishmaniasis in absence of treatment. Genome instability has been recognized as a driver in Leishmania fitness gain in response to environmental change or chemotherapy. How genome instability generates beneficial phenotypes despite potential deleterious gene dosage effects is unknown. Here we address this important open question applying experimental evolution and integrative systems approaches on parasites adapting to in vitro culture. Phenotypic analyses of parasites from early and late stages of culture adaptation revealed an important fitness tradeoff, with selection for accelerated growth in promastigote culture (fitness gain) impairing infectivity (fitness costs). Comparative genomics, transcriptomics and proteomics analyses revealed a complex regulatory network associated with parasite fitness gain, with genome instability causing highly reproducible, gene dosage-independent and -dependent changes. Reduction of flagellar transcripts and increase in coding and non-coding RNAs implicated in ribosomal biogenesis and protein translation were not correlated to dosage changes of the corresponding genes, revealing a gene dosage-independent, post-transcriptional mechanism of regulation. In contrast, abundance of gene products implicated in post-transcriptional regulation itself correlated to corresponding gene dosage changes. Thus, RNA abundance during parasite adaptation is controled by direct and indirect gene dosage changes. We correlated differential expression of small nucleolar RNAs (snoRNAs) with changes in rRNA modification, providing first evidence that Leishmania fitness gain in culture may be controlled by post-transcriptional and epitranscriptomic regulation. Our findings propose a novel model for Leishmania fitness gain in culture, where differential regulation of mRNA stability and the generation of modified ribosomes may potentially filter deleterious from beneficial gene dosage effects and provide proteomic robustness to genetically heterogenous, adapting parasite populations. This model challenges the current, genome-centric approach to Leishmania epidemiology and identifies the Leishmania transcriptome and non-coding small RNome as potential novel sources for the discovery of biomarkers that may be associated with parasite phenotypic adaptation in clinical settings.

Published
2022
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48. Insights into amebiasis using a human 3D-intestinal model

Author

Nancy Guillén, Mariette Matondo, Julien Fernandes, Jean-Yves Coppée, Rachel Legendre, Silvia Castellanos-Castro, Hugo Varet, David Hardy, Odile Sismeiro, Arturo Aguilar-Rojas, Quentin Giai Gianetto, Jean-Christophe Olivo-Marin, Analyse d'images biologiques - Biological Image Analysis (BIA), Institut Pasteur [Paris]-Centre National de la Recherche Scientifique (CNRS), Instituto Mexicano del Seguro Social [Mexico City, Mexico] (IMSS), Universidad Autónoma de la Ciudad de México (UACM), Spectrométrie de Masse pour la Biologie – Mass Spectrometry for Biology (UTechS MSBio), Institut Pasteur [Paris]-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS), Transcriptome et Epigénome (PF2), Institut Pasteur [Paris], Hub Bioinformatique et Biostatistique - Bioinformatics and Biostatistics HUB, BioImagerie Photonique – Photonic BioImaging (UTechS PBI), Neuropathologie expérimentale / Experimental neuropathology, Institut Pasteur [Paris]-Université de Paris (UP), The project has received funding from the European ERA‐NET Infect‐ERA program AMOEBAC (French National Agency for Research [ANR] grants ANR‐14‐IFEC‐0001‐01 and ANR‐14‐IFEC‐0001‐02). Biomics Platform (Institute Pasteur) is supported by France Génomique (ANR‐10‐INBS‐09‐09) and IBISA. AAR is a recipient of Research Career Development Awards from Fundación‐IMSS, México. SCC received a postdoctoral fellowship with number 173697 from the program S190‐Conacyt, 2017, Mexico., ANR-14-IFEC-0001,AMOEBAC,Entamoeba histolytica-bacterium interaction and the role of this interaction in intestinal pathogenesis.(2014), ANR-10-INBS-0009,France-Génomique,Organisation et montée en puissance d'une Infrastructure Nationale de Génomique(2010), Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS), Institut Pasteur [Paris] (IP)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS), Institut Pasteur [Paris] (IP), and Institut Pasteur [Paris] (IP)-Université Paris Cité (UPCité)

Subjects
Models, Anatomic, Immunology, Virulence, Inflammation, Microbiology, Host-Parasite Interactions, Adherens junction, 03 medical and health sciences, Entamoeba histolytica, Immune system, Virology, medicine, Humans, [SDV.MP.PAR]Life Sciences [q-bio]/Microbiology and Parasitology/Parasitology, Fibroblast, 030304 developmental biology, 0303 health sciences, Microscopy, Confocal, biology, 030306 microbiology, Amebiasis, biology.organism_classification, In vitro, 3. Good health, Intestines, secretome, medicine.anatomical_structure, 3D-intestinal model, Infectious disease (medical specialty), inflammation, Dysentery, Amebic, medicine.symptom, transcriptome
Abstract

International audience; Entamoeba histolytica is the causative agent of amebiasis, an infectious disease targeting the intestine and the liver in humans. Two types of intestinal infection are caused by this parasite: silent infection, which occurs in the majority of cases, and invasive disease, which affects 10% of infected persons. To understand the intestinal pathogenic process, several in vitro models, such as cell cultures, human tissue explants or human intestine xenografts in mice, have been employed. Nevertheless, our knowledge on the early steps of amebic intestinal infection and the molecules involved during human-parasite interaction is scarce, in part due to limitations in the experimental settings. In the present work, we took advantage of tissue engineering approaches to build a three-dimensional (3D)-intestinal model that is able to replicate the general characteristics of the human colon. This system consists of an epithelial layer that develops tight and adherens junctions, a mucus layer and a lamina propria-like compartment made up of collagen containing macrophages and fibroblast. By means of microscopy imaging, omics assays and the evaluation of immune responses, we show a very dynamic interaction between E. histolytica and the 3D-intestinal model. Our data highlight the importance of several virulence markers occurring in patients or in experimental models, but they also demonstrate the involvement of under described molecules and regulatory factors in the amoebic invasive process.

Published
2020
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49. Impact of the gut microbiota on the m 6 A epitranscriptome of mouse cecum and liver

Author

Pascale Cossart, Marie-Agnès Dillies, Sabrina Jabs, Amine Ghozlane, Quentin Giai Gianetto, Vincent Guérineau, Christophe Bécavin, David Touboul, Alessandro Pagliuso, Giulia Spanò, Mariette Matondo, Marie-Anne Nahori, Thibault Chaze, Anne Biton, Interactions Bactéries-Cellules (UIBC), Institut National de la Recherche Agronomique (INRA)-Institut Pasteur [Paris] (IP)-Institut National de la Santé et de la Recherche Médicale (INSERM), Hub Bioinformatique et Biostatistique - Bioinformatics and Biostatistics HUB, Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS), Institut de Chimie des Substances Naturelles (ICSN), Institut de Chimie du CNRS (INC)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS), Spectrométrie de Masse pour la Biologie – Mass Spectrometry for Biology (UTechS MSBio), Institut Pasteur [Paris] (IP)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS), We thank Marion Bérard and the Gnotobiology platform of the Institut Pasteur for support in conducting gnotobiology experiments, the Pasteur Biomics platforms for Transcriptomics and Epigenetics and Metagenomics for providing RNA sequencing and 16S rRNA sequencing services, Cédric Fund, Biomics Platform, C2RT, Institut Pasteur, Paris, France, supported by France Génomique (ANR-10-INBS-09-09) and IBISA, Carla Saleh and Olivier Dussurget for helpful discussions, Hugo Varet (Institut Pasteur Bionformatics and Statistics Hub) for advice concerning statistical analysis, Stevenn Volant for help with analysis of 16S sequencing data. This work was supported by grants to P.C.: European Research Council (ERC) Advanced Grant BacCellEpi (670823), ANR Investissement d’Avenir Programme (10-LABX-62-IBEID), ERANET-Infect-ERA PROANTILIS (ANR-13-IFEC-0004-02) and the Fondation Le Roch-Les Mousquetaires. S.J. was supported by a Danone Research fellowship and European Research Council (ERC) Advanced Grant BacCellEpi (670823)., ANR-10-INBS-0009,France-Génomique,Organisation et montée en puissance d'une Infrastructure Nationale de Génomique(2010), ANR-10-LABX-0062,IBEID,Integrative Biology of Emerging Infectious Diseases(2010), ANR-13-IFEC-0004,PROANTILIS,Subversive pro- and anti-inflammation trade-offs promote infection by Listeria monocytogenes(2013), European Project: 670823,H2020,ERC-2014-ADG,BacCellEpi(2015), Nahori, marie-anne, Organisation et montée en puissance d'une Infrastructure Nationale de Génomique - - France-Génomique2010 - ANR-10-INBS-0009 - INBS - VALID, Integrative Biology of Emerging Infectious Diseases - - IBEID2010 - ANR-10-LABX-0062 - LABX - VALID, ERA-NET Infect-ERA - Subversive pro- and anti-inflammation trade-offs promote infection by Listeria monocytogenes - - PROANTILIS2013 - ANR-13-IFEC-0004 - IFEC - VALID, Bacterial, cellular and epigenetic factors that control enteropathogenicity - BacCellEpi - - H20202015-10-01 - 2018-09-30 - 670823 - VALID, Institut National de la Recherche Agronomique (INRA)-Institut Pasteur [Paris]-Institut National de la Santé et de la Recherche Médicale (INSERM), Institut Pasteur [Paris]-Centre National de la Recherche Scientifique (CNRS), and Institut Pasteur [Paris]-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS)

Subjects
0301 basic medicine, Adenosine, Methyltransferase, Science, [SDV]Life Sciences [q-bio], General Physics and Astronomy, Biology, Gut flora, Methylation, digestive system, Article, General Biochemistry, Genetics and Molecular Biology, Microbiology, Transcriptome, Mice, 03 medical and health sciences, Cecum, 0302 clinical medicine, fluids and secretions, Gene expression, medicine, Animals, RNA, Messenger, lcsh:Science, Multidisciplinary, Bacteria, Methyltransferases, General Chemistry, biology.organism_classification, Commensalism, Gastrointestinal Microbiome, Mice, Inbred C57BL, [SDV] Life Sciences [q-bio], stomatognathic diseases, 030104 developmental biology, medicine.anatomical_structure, Liver, Female, lcsh:Q, Microbiome, 030217 neurology & neurosurgery, Akkermansia muciniphila
Abstract

The intestinal microbiota modulates host physiology and gene expression via mechanisms that are not fully understood. Here we examine whether host epitranscriptomic marks are affected by the gut microbiota. We use methylated RNA-immunoprecipitation and sequencing (MeRIP-seq) to identify N6-methyladenosine (m6A) modifications in mRNA of mice carrying conventional, modified, or no microbiota. We find that variations in the gut microbiota correlate with m6A modifications in the cecum, and to a lesser extent in the liver, affecting pathways related to metabolism, inflammation and antimicrobial responses. We analyze expression levels of several known writer and eraser enzymes, and find that the methyltransferase Mettl16 is downregulated in absence of a microbiota, and one of its target mRNAs, encoding S-adenosylmethionine synthase Mat2a, is less methylated. We furthermore show that Akkermansia muciniphila and Lactobacillus plantarum affect specific m6A modifications in mono-associated mice. Our results highlight epitranscriptomic modifications as an additional level of interaction between commensal bacteria and their host., The intestinal microbiota modulates host physiology and gene expression via unclear mechanisms. Here, Jabs et al. show that variations in the gut microbiota correlate with N6-methyladenosine modifications of host mRNAs in the cecum and liver of mice.

Published
2020
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50. Phosphorylation-Dependent Assembly of a 14-3-3 Mediated Signaling Complex During Red Blood Cell Invasion by Plasmodium falciparum Merozoites

Author

Jyoti S. Choudhary, Brandon M. Invergo, Christèle Huon, Petra Gutenbrunner, Mariette Matondo, Inderjeet Kaur, Kunal R. More, Chetan E. Chitnis, Shailja Singh, Ravi Jain, Hendrik Weisser, Thibault Chaze, Gordon Langsley, Quentin Giai Gianetto, Biologie de Plasmodium et Vaccins - Malaria Parasite Biology and Vaccines, Institut Pasteur [Paris], International Centre for Genetic Engineering and Biotechnology [New Delhi] (ICGEB), Spectrométrie de Masse pour la Biologie – Mass Spectrometry for Biology (UTechS MSBio), Institut Pasteur [Paris]-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS), Hub Bioinformatique et Biostatistique - Bioinformatics and Biostatistics HUB, Institut Pasteur [Paris]-Centre National de la Recherche Scientifique (CNRS), European Bioinformatics Institute [Hinxton] (EMBL-EBI), EMBL Heidelberg, The Wellcome Trust Sanger Institute [Cambridge], Jawaharlal Nehru University (JNU), Biologie cellulaire comparative des Apicomplexes, [Institut Cochin] Departement Infection, immunité, inflammation, Institut Cochin (IC UM3 (UMR 8104 / U1016)), Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Université de Paris (UP)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Université de Paris (UP)-Institut Cochin (IC UM3 (UMR 8104 / U1016)), Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Université de Paris (UP)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Université de Paris (UP), This work was supported by a MolSigMal grant (ANR-17-CE15-0010) from Agence Nationale de la Recherche (ANR) to C.E.C. and internal funds from the Institut Pasteur. G.L. acknowledges support from the Labex ParaFrap (ANR-11-LABX-0024). K.R.M. received a short-term fellowship from the European Molecular Biology Organization (EMBO)., ANR-17-CE15-0010,MolSigMal,Evènements moléculaires de la signalisation induite par l'invasion des globules rouges par les parasites paludéens(2017), ANR-11-LABX-0024,ParaFrap,Alliance française contre les maladies parasitaires(2011), Institut Pasteur [Paris] (IP), Institut Pasteur [Paris] (IP)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS), Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS), Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Université Paris Cité (UPCité)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Université Paris Cité (UPCité)-Institut Cochin (IC UM3 (UMR 8104 / U1016)), Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Université Paris Cité (UPCité)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Université Paris Cité (UPCité), PORTELETTE, Virginie, Evènements moléculaires de la signalisation induite par l'invasion des globules rouges par les parasites paludéens - - MolSigMal2017 - ANR-17-CE15-0010 - AAPG2017 - VALID, and Laboratoires d'excellence - Alliance française contre les maladies parasitaires - - ParaFrap2011 - ANR-11-LABX-0024 - LABX - VALID

Subjects
Proteomics, Erythrocytes, Multiprotein complex, [SDV.BBM.BS] Life Sciences [q-bio]/Biochemistry, Molecular Biology/Structural Biology [q-bio.BM], Plasmodium falciparum, Protozoan Proteins, malaria, Microbiology, Host-Microbe Biology, Microneme, 03 medical and health sciences, 0302 clinical medicine, hemic and lymphatic diseases, Virology, parasitic diseases, Humans, Protein phosphorylation, Phosphorylation, Protein kinase A, host cell invasion, 030304 developmental biology, 0303 health sciences, biology, [SDV.BBM.BS]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Structural Biology [q-bio.BM], Merozoites, Kinase, Chemistry, Phosphoproteomics, hemic and immune systems, biology.organism_classification, host-parasite interaction, QR1-502, 3. Good health, Cell biology, 14-3-3 Proteins, Signal transduction, signaling, 030217 neurology & neurosurgery, circulatory and respiratory physiology, Signal Transduction, Research Article
Abstract

Invasion of red blood cells (RBCs) by Plasmodium falciparum merozoites is a complex process that is regulated by intricate signaling pathways. Here, we used phosphoproteomic profiling to identify the key proteins involved in signaling events during invasion. We found changes in the phosphorylation of various merozoite proteins, including multiple kinases previously implicated in the process of invasion. We also found that a phosphorylation-dependent multiprotein complex including signaling kinases assembles during the process of invasion. Disruption of this multiprotein complex impairs merozoite invasion of RBCs, providing a novel approach for the development of inhibitors to block the growth of blood-stage malaria parasites., Red blood cell (RBC) invasion by Plasmodium merozoites requires multiple steps that are regulated by signaling pathways. Exposure of P. falciparum merozoites to the physiological signal of low K+, as found in blood plasma, leads to a rise in cytosolic Ca2+, which mediates microneme secretion, motility, and invasion. We have used global phosphoproteomic analysis of merozoites to identify signaling pathways that are activated during invasion. Using quantitative phosphoproteomics, we found 394 protein phosphorylation site changes in merozoites subjected to different ionic environments (high K+/low K+), 143 of which were Ca2+ dependent. These included a number of signaling proteins such as catalytic and regulatory subunits of protein kinase A (PfPKAc and PfPKAr) and calcium-dependent protein kinase 1 (PfCDPK1). Proteins of the 14-3-3 family interact with phosphorylated target proteins to assemble signaling complexes. Here, using coimmunoprecipitation and gel filtration chromatography, we demonstrate that Pf14-3-3I binds phosphorylated PfPKAr and PfCDPK1 to mediate the assembly of a multiprotein complex in P. falciparum merozoites. A phospho-peptide, P1, based on the Ca2+-dependent phosphosites of PKAr, binds Pf14-3-3I and disrupts assembly of the Pf14-3-3I-mediated multiprotein complex. Disruption of the multiprotein complex with P1 inhibits microneme secretion and RBC invasion. This study thus identifies a novel signaling complex that plays a key role in merozoite invasion of RBCs. Disruption of this signaling complex could serve as a novel approach to inhibit blood-stage growth of malaria parasites.

Published
2020
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